Cuticular waxes are complex mixtures of very long chain fatty acids (VLCFAs) and their derivatives. The CER2 locus of Arabidopsis is involved in cuticular wax accumulation on stems, siliques, and hypocotyls. The CER2 gene was cloned via chromosome walking. This cloned sequence is able to complement the cer2 mutant phenotype. It is a single-copy sequence in the Arabidopsis genome and encodes a novel protein with a predicted mass of 47 kD. Anti-CER2 polyclonal antibodies detected a 47 kD polypeptide. Cell fractionation and immunoblot analyses demonstrated that the CER2 protein is not membrane-bound and localized in nuclei. These results suggest that CER2 might have a regulatory role in cuticular wax accumulation. The expression patterns of the CER2 gene were studied by in situ RNA hybridization and the analysis of Arabidopsis transgenic plants harboring an in-frame fusion of -1009/+234 of the CER2 gene to [beta]-glucuronidase (CER2-GUS). These analysis demonstrate that CER2 gene expression is developmentally regulated and organ- and tissue-specific. Consistence with the visible phenotype associated with cer2 mutants, the CER2 gene is highly expressed only on the epidermal cells of stems, siliques, and hypocotyls. In addition, CER2 expression was observed in guard cells, trichomes, petioles, sepals, petals, ovaries, pedicels, the tapetum layer of anthers, and pollen grains. CER2-GUS expression was not detected in roots or in the pavement cells of leaves. The observation of CER2 expression in anthers and pollen grains is in agreement with the fact that the CER2 gene is involved in pollen fertility. Light, drought, high osmotic pressure, heat or cold shock, and wounding were not be found to cause visible changes in CER2-GUS expression patterns. However, exogenous application of the cytokinin, BAP, induced ectopic expression of CER2-GUS in leaves, suggesting that CER2 gene expression might be mediated by endogenous cytokinins.