1. 1. Soluble extracts of rat ventral prostate contain a calcium-dependent, neutral thiol protease which is separated from an endogenous inhibitor by DEAE-cellulose chromatography. 2. 2. The Ca2+-dependent protease had a high calcium requirement (half maximal activation at 0.19mM CaCl2). a pH optimum in the neutral range (pH 7-8), and it was inhibited by increased ionic strength (30% inhibition at 0.2 M NaCl). 3. 3. Leupeptin and antipain were strong inhibitors of the enzyme. Ca2+-activated protease activities of the coagulating gland (anterior prostate) were about 40% of those of the ventral prostate and were not detectable in the dorsolateral prostatic lobe. 4. 4. There was no difference in specific activities of this enzyme in chromatographed extracts of prostatic lobes from young sexually mature adults and 12 month old retired breeders. 5. 5. In addition, Ca2+-dependent protease activity was not detectable in chromatograms of rat ventral prostate and coagulating gland secretions. 6. 6. Therefore, the Ca2+-activated protease does not appear to be a secretory protein and probably acts at some intracellular site(s).