Aminopeptidases (APN) may play a role in host colonization of M. gallinarum. Characterization of endogenous APN activity suggests that the leucine APN (LAP) of M. gallinarum is a metallo-aminopeptidase activated by Mn2+ and is present in the cytosol and possibly associated with the inner leaflet of the membrane. A 1.36-kb open reading frame (ORF) identified from overlapping genomic phage clones showed 68% nucleotide identity and 51% amino acid identity with the M. salivarium LAP gene. This ORF is expressed as a 1.5-kb monocistronic transcript and is present as a single copy in M. gallinarum. This gene sequence was modified to account for codon usage, and expression in E. coli produced a 51-kDa protein, which compares well with the product predicted from the ORE This ORF is a strong candidate for contributing the LAP activity of M. gallinarum protein extracts.