The acrosomal proteinase of human spermatozoa was characterized and differs from other human proteinases. The enzyme has optimal activity at p H 8.0, is inactive below p H 5.0 or above p H 10.5, requires calcium for maximum activity, hydrolyzes fibrinogen, gelatin, and benzoylarginine ethyl ester, and is inhibited by various synthetic and natural proteinase inhibitors. The molecular weight was estimated to be 30,000. Human spermatozoa also possess a proteinase inhibitor that is similar to one of the proteinase inhibitors from human seminal plasma.