A comparison of the alkaline proteinase activity of human seminal plasma, the seminal non-gamete cellular material and spermatozoa was made by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (gelatin-SDS-PAGE) zymography. Several major (molecular weights = greater than 56,000) and minor (35,000 to 44,000) bands of proteinase activity were seen in the seminal plasma samples from nonvasectomized and vasectomized, healthy donors. Similar activity profiles were observed in the nongamete cellular material of vasectomized donor ejaculates. The major proteinase activity in sperm extracts was in the 47,000 to 55,000 (proacrosin-acrosin) and 34,000 to 37,000 (sperminogen-spermin) molecular weight ranges. These results suggest that the proacrosin-acrosin and sperminogen-spermin systems are of sperm origin and that there are considerable amounts of larger molecular weight trypsin-like enzymes in the soluble and nongamete cellular material of human seminal plasma.