Mycoplasmas are deficient in numerous biosynthetic pathways. Consequently, growth is dependent upon the acquisition of structural and macromolecular precursors from the environment. These precursors include phospholipids, fatty acids and nucleotide bases. DNases and RNases are postulated to be involved in the acquisition of nucleotides. The goals of this study were to determine if nuclease activities are universally associated with mycoplasmas, characterize the nucleases by subcellular distribution and apparent molecular weight, and to identify and analyze the nuclease gene(s) in Mycoplasma pulmonis;Nuclease activity was present in all mycoplasma species tested. The level of activity did not correlate with virulence or growth rates, however. The cation preference for the nucleases varied among the species. In most cases, treatment of the cells with detergents enhanced the activity. All of the nuclease activity was localized in the mycoplasma membrane. The nucleolytic protein SDS-PAGE profiles of each species were distinctive, with most species producing more than one nucleolytic protein. Analysis of multiple strains demonstrated that the nucleolytic banding profiles may be useful as classification tools;In order to further define membrane nucleases of M. pulmonis, a genomic library was screened to identify nuclease positive recombinant phage. Chromosomal fragments in four nuclease positive phage were restriction mapped and subcloned. The inserts, representing two different regions of the M. pulmonis chromosome, contained at least three nuclease genes. Only one of the fragments demonstrated stable expression of activity when subcloned into a plasmid vector. Mutagenesis with Tn1000 was used to localize the nuclease gene in this fragment and to facilitate DNA sequencing. An open reading frame of 1410 bp was identified which coded for a protein of 53.7 kDa. The presence of a membrane spanning domain and the prolipoprotein signal sequence TISC indicated that the protein is a membrane lipoprotein. This gene has been designated mnuA for membrane nuclease;The mnuA gene of M. pulmonis was part of a two gene-operon which also contained an uvrB homolog. This uvrB homolog shared 51% identity and 70% homology with uvrB of Haemophilus influenzae and was located upstream of mnuA.