Insect development, metamorphosis and reproduction are regulated in part by the action of juvenile hormone (JH). The titer of JH is regulated in turn by the action of the enzymes juvenile hormone epoxide hydrolase and juvenile hormone esterase (JHE). Because of the potential for disruption of regulation of insect development through perturbation of the action of JH, the biology of JHE has been well studied. A putative juvenile hormone esterase binding protein, P29 was identified in the tobacco hornworm, Manduca sexta. Following sequencing of the Drosophila melanogaster genome, we identified a homolog of P29 in D. melanogaster, and used this insect for analysis of the biology and function of P29 in relation to JHE.;The gene encoding D. melanogaster P29 (DmP29), CG3776 was cloned, recombinant DmP29 expressed in E. coli and two anti-DmP29 antisera raised. In vitro binding of the P29 homolog to Drosophila JHE was confirmed. P29 mRNA and an immunoreactive protein of 25 kDa were detected in Drosophila larvae, pupae and adults. The predicted size of the protein is 30kD. Drosophila P29 is predicted to localize to mitochondria (MitoProt; 93% probability) and has a 6kD N-terminal targeting sequence. Subcellular organelle fractionation and confocal microscopy of Drosophila S2 cells confirmed that the immunoreactive 25kD protein is present in mitochondria but not in the cytosol. Expression of P29 without the predicted N-terminal targeting sequence in High Five(TM) cells showed that the N-terminal targeting sequence is shorter than predicted, and that a second, internal mitochondrial targeting signal is also present. An immunoreactive protein of 50 kDa in the hemolymph does not result from alternative splicing of CG3776 but may result from dimerization of P29.;We investigated the potential ligands of DmP29 by testing three hypotheses: (i) DmP29 binds to D. melanogaster JHE: We produced a stably transformed insect cell line that expresses DmJHE and confirmed that DmP29 binds to D. melanogaster P29. DmJHE binds to both the 25 kD and 50 kD immunoreactive proteins. (ii) DmP29 binds other, non-specific esterases including two esterases predicted to be targeted to the mitochondria: We did not detect any interaction between DmP29 and non-specific esterases. (iii) DmP29 binds to other proteins in D. melanogaster: Ligand blot analysis, immunoprecipitation experiments and affinity binding experiments showed that larval serum protein 1 binds the 25 kD P29. The possible biological relevance of the in vitro DmP29-JHE interaction is provided by detection of JHE activity in D. melanogaster mitochondrial fractions; 0.48 nmol JH hydrolysed/min/mg mitochondrial protein, 97% of which was inhibited by the JHE-specific inhibitor OTFP. However, the DmP29-LSP interactions may not be biologically relevant, given the high abundance, and "sticky" nature of these proteins. Interaction of DmP29 with LSP may result from non-specific associations. We used P29 hypo- and hyper-expression mutants to elucidate the function of P29 and the potential interaction of P29 with JHE. The hypomorphic mutant EP835 of P29 had reduced JHE activity when compared to wild type flies. Hyperexpression of P29 in EP/Gal4 during the early larval stages was lethal, while hyperexpression during the third instar resulted in reduced size of adult flies. This phenotype showed that overexpression of P29 interfered with insect development. Hyperexpression in newly eclosed but not in older females resulted in reduced fecundity, indicating that overexpression of P29 affected ovarian development. Fecundity was not affected by P29 hyperexpression in the male. Hypermorphic adults exhibited male-male courtship behavior. Hyperexpressed females showed reduced receptivity to males. Hyperexpressed females had decreased production of courtship pheromone, cis, cis-7, 11-hepta cosadiene, which resulted in male flies being unable to locate female flies. Hyperexpression of P29 in males resulted in decreased production of the aggregation pheromone, cis-vaccenyl acetate. For EP835/Gal4, the hypermorphic mutant, all hyperexpression phenotypes were consistent with a reduced JH titer in Drosophila. Flies that hypo- or hyper-expressed P29 had a significantly shorter lifespan: Reduced lifespan correlated with increased egg production (hypomorphic flies) and hyperactivity (hypermorphic flies), respectively. Hence, the titer of P29 appeared to be positively correlated with the titer of JHE and negatively correlated with the titer of JH. Based on the collective phenotypes and detection of JHE activity in mitochondria, we hypothesize that JHE is stored in mitochondria and that P29 functions in transport of JHE to the cytosol.