Aminopeptidases catalyze the cleavage of specific amino acids from the amino terminus of protein or peptide substrates. A proline-specific aminopeptidase was purified to homogeneity from the culture-free extract of Streptomyces lavendulae ATCC 14162 in sequential steps comprising ammonium sulfate precipitation, ultra-filtration, and column chromatography on Q-sepharose and Sephadex G-100. The purified protein showed approximately 60 kDa in SDS-PAGE and was optimally active at pH 6.5 and 40 A degrees C. Kinetic studies showed a K (m) and V (max) of 0.23 mM and 0.087 mu mol/min, respectively, using Pro-p-NA, the substrate with maximum specificity. Enzyme activity was inhibited by PMSF and ions like Zn2+, Co2+, and Ni2+. However, unlike other aminopeptidases, the activity was enhanced in the presence of DTT, 1,10-phenanthroline, EDTA, amastatin, and bestatin. Ions like Ca2+, Mg2+, and Mn2+ also enhanced the activity.