AbstractA proteolytic enzyme capable of cleaving intact proteins and synthetic substrates α−N−benzoyl−DL−arginine β−naphthylamide (Bz‐Arg‐NNap), α‐N‐benzoyl‐L‐arginine p‐nitroanilde (Bz‐Arg‐NPhNO2), and α‐N‐benzoyl‐L‐arginine ethyl ester (Bz‐Arg‐OEt) was purified 92– fold from the rabbit testes. The enzyme exhibited optimal activity at pH 9.0 and 50°C. The polyacrylamide gel electrophoresis and sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis of the purified enzyme demonstrated multiple forms; the major band in the SDS‐polyacrylamide gel electrophoresis corresponded to a Mt48,000. The same value was established by the gel filtration over Sephadex G‐75. The rabbit testicular alkaline proteinase (TAP) resembled acrosin in the hydrolysis of Bz‐Arg‐OEt. However, CaCl2, a potential stimulator of acrosin activity, inhibited the alkaline proteinase. The strong inhibitors of acrosin, eg pheny methyl sulphonyl fluoride (PMSF), tosyl lysine chloromethyl ketone (TLCK), and benzamidine did not inhibit the alkaline proteinase. TAP was activated by an acrosin inhibitor isolated from the rabbit testes. Since 0.5 M KCl was necessary for complete extraction of the enzyme and the bulk of the activity was present in 9,000g pellet of the testicular homogenate. The alkaline proteinase