The mechanism responsible for the spontaneous initiation of proacrosin conversion into acrosin in vitro was studied and characterized by using the highly effective inhibitor leupeptin. In the presence of excess leupeptin [10(2)-10(3))KI to acrosin], proacrosin spontaneously and completely converted into acrosin at pH 8. However, only the initial enzyme product, m alpha-acrosin, was produced, and the rate of conversion was not affected when exogenous m alpha-acrosin was added to the reaction mixture. These results demonstrate that excess leupeptin eliminated all conversion and degradative reactions requiring active acrosin. Kinetically, the conversion of proacrosin into m alpha-acrosin in the presence of excess leupeptin appeared first order. The observed half-life (t 1/2 = 1.4 h) did not vary over a 10-fold range of leupeptin or initial proacrosin concentrations. These data demonstrate that proacrosin can self-catalyze its own conversion into m alpha-acrosin by an intrazymogen mechanism.