The preparation of highly purified malpha-acrosin is described. Purification was achieved by controlled activation of partially purified proacrosin, followed by gel chromatography over Sephadex G-100 at pH 3.0. The final malpha-acrosin preparation resulted in a single protein band with a molecular weight of 49,000 as determined by sodium dodecyl sulfate-disc gel electrophoresis. Disc arginine naphthylamide hydrolyzing band with a relative migration of 0.39 malpha-acrosin catalyzed the hydrolysis of synthetic substrates containing arginine and lysine, but not phenylalanine. Although calcium ions were not required for enzymatic activity, the addition of calcium chloride stimulated the activity through an increased substrate affinity and an increased maximal velocity. Polyamines stimulated the maximal velocity of the reaction, but were without effect on the substrate affinity. malpha-Acrosin was inhibited by lima bean, ovo-mucoid, and seminal plasma proteinase inhibitors. Diisopropyl fluorophosphate and 1-chloro-3-tosylamide-7-amino-L-2-heptanone treatment resulted in an irreversible inhibition, while L-arginine, benzamidine, and p-aminobenzamidine were competitive inhibitors with respect to substrate. These properties of malpha-acrosin are very similar to those previously reported for mbeta-acrosin and suggest that the portion of the molecule lost during the conversion of malpha-acrosin to mbeta-acrosin contributes little to the topography of either the active site or regulatory sites of the enzyme.