Guinea pig epididymal spermatozoa were either capacitated only, or capacitated and acrosome-reacted in vitro. The spermatozoa were separated from the supernatant incubation media, the spermatozoa extracted and the amount of acrosin [EC 3.4.21.10], proacrosin and acrosin inhibitor in the sperm extracts and in the supernatant solutions was measured. A dialyzable factor was present which inhibited proacrosin activation and acrosin activity, although the latter to a lesser degree, and was partially removed during the acrosome reaction. The total amount of acrosin (nonzymogen acrosin + proacrosin) in untreated, control spermatozoa was 3-5 times that in mouse and human spermatozoa. A reduction of .apprx. 50% in the total amount of sperm acrosin occurred after induction of the acrosome reaction. Whether spermatozoa were untreated, capacitated or acrosome-reacted, .apprx. 90% of the acrosin present on the spermatozoa was in proacrosin form. The loss of acrosin activity after induction of the acrosome reaction was due to its release into the surrounding medium, and all of the released enzyme was in the nonzymogen acrosin form. The amount of acrosin released from control spermatozoa kept in buffer, which underwent the false acrosome reaction, was approximately the same as that released from true acrosome-reacted spermatozoa. The total amount of acrosin released after induction of the acrosome reaction was never more than about 55% of the original amount present, even if more than 80% of the spermatozoa had undergone the acrosome reaction. Proacrosin activation apparently does not occur, or occurs only minimally, during in vitro capacitation of guinea pig spermatozoa, but approximately 1/2 is activated just before, during or immediately after the spermatozoa show morphological changes in the acrosome, whether induced by sperm death or capacitation. Approximately the same small amount of acrosin inhibitor was released from capacitated spermatozoa as from noncapacitated spermatozoa kept in buffer that had undergone the false acrosome reaction. A much higher amount of acrosin inhibitor was liberated from capacitated, acrosome-reacted spermatozoa.