The ventral, lateral, and dorsal lobes of the rat prostate produce secretions rich in protein. We examined these secretions for proteinase activities, using gelatin- and casein-containing SDS-polyacrylamide gel zymographies. The ventral lobe demonstrated both higher activities and more molecular forms of proteinase activities that cleaved these two protein substrates than did the other lobes. Ca2+-stimulated gelatinolytic activities of approximately 48, 51, 58, 64, 74, and 80 kDa were found in ventral prostate secretions in addition to activities detected in the absence of added Ca2+: a very strong 27-kDa activity; prominent 22-, 86-, 90-, and 94-kDa activities; and less active 36-, 41-, 100-, 130-, and 140-kDa activities. Ca2+-stimulated gelatinolytic activities of 51, 58, 74, 80, 86, 90, and 94 kDa were present in lateral prostate secretions (none were detected in secretions of the dorsal lobe), and Ca2+-independent activities of 25, 27, and 100 kDa were found in secretions of both the lateral and dorsal lobes. The Ca2+-stimulated activities were inhibited by EGTA and EDTA. Benzamidine inhibited all gelatinolytic activities except for the 22-, 25-, and 27-kDa Ca2+-independent forms when Ca2+ was not added to the reaction buffer. However, in the presence of 5 mM CaCl2, the Ca2+- stimulated forms of proteinase were unaffected by benzamidine, whereas the other activities sensitive to benzamidine were inhibited. Prominent Ca2+- independent caseinolytic activities of 20, 23, 31, 37, 83, 89, and 94 kDa were detected in ventral lobe secretions along with less active forms of about 39, 48, 53, 57, 60, 63, 80, 103, 110, 125, and 160 kDa. Caseinolytic activities of approximately 23, 31, 53, 89, 94, 103, 120, and 125 kDa were found in lateral prostate secretions, and 89, 94, and 103 kDa activities were found in secretions of the dorsal lobe. Quantitatively, most gelatinolytic and caseinolytic activities were present in the soluble portion of the secretion of each prostatic lobe. The ventral, lateral, and dorsal prostate lobes all secrete gelatinolytic and caseinolytic proteinase activities; however, quantitatively the ventral lobe is the most notable in this function since its secretions contain more molecular forms and greater activities of these proteinases.