Two different forms of oxytocinase (L-cystine aminopeptidase, CAP; EC 3.4.11.3) were purified from the 9000 g and 105000 g precipitate fractions of human placenta homogenate by sequential chromatography on columns of hydroxyapatite, DE-32, nickel ion affinity, and Sephadex G-200. One species (CAP-I) purifed from the mitochondrial/lysosomal fraction migrated on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with an apparent molecular mass of 61 kDa; the other (CAP-II) from the microsomal fraction was composed of two subunits with molecular masses of 56 and 40 kDa. The molecular masses of CAP-I and CAP-II estimated by gel filtration were 64 and 97 kDa, respectively. The specific activities of the two species for S-benzyl-L-cysteine p-nitroanilide increased by 357- (for CAP-I) and 139-fold (for CAP-II) compared with the starting preparations. The optimal pH values toward the artificial substrate were approx. 7.4-8.0 for CAP-I and 6.8-8.0 for CAP-II. The Km and Vmax values toward oxytocin were 5.6 microM and 23.4 micromol/h/mg protein for CAP-I, and 38 microM and 15.6 micromol/h/mg protein for CAP-II. Both enzymes were inhibited by the metal-chelating agents, EDTA and o-phenanthroline, whereas they were specifically activated by addition of Co2+: CAP-I was more sensitive to these reagents than CAP-II. L-Methionine strongly inhibited CAP-I, while CAP-II activity was only slightly affected. CAP-II was more sensitive to amastatin than CAP-I. Thus, the two enzymes are quite distinct in their molecular nature and biochemical properties. They may play a regulatory role in the metabolism of oxytocin and other biologically active peptides in intact placenta.