Based on recent studies indicating that human glandular kallikrein-1 (hGK-1) mRNA was present in the prostate, we have undertaken to determine whether the prostate contained trypsin-like proteases with properties compatible with those deduced from hGK-1 gene nucleotide sequence. The first series of experiments showed that only minimal levels of trypsin-like enzymatic activity, determined with synthetic substrates, were present in chromatographic fractions of prostatic glycoproteins having a molecular weight in the range expected for hGK-1, i.e., 25,000-35,000. Because of this, we used [3H]diisopropylfluorophosphate (DFP) labeling alone or in the presence of various serine-protease inhibitors to identify trypsin-like proteases in the prostate. Two-dimensional gel electrophoresis of prostatic glycoproteins showed the presence of minor spots of 18-32 kDa. These spots were slightly more acidic than were those of prostate specific antigen (PSA) and were completely inhibited by preincubation with tosyl lysine chloromethyl ketone and p-nitrophenyl-p-guanidobenzoate in contrast to PSA spots that were not affected by these treatments. When a similar procedure was applied to total cytosolic proteins instead of glycoproteins, an additional 30 kDa DFP binding protein was observed. This relatively abundant protein was much more acidic than was PSA and was not inhibited by any of the protease inhibitors tested. In conclusion, this study has permitted us to demonstrate the presence of two sets of proteins that have physicochemical properties compatible with those that can be deduced from the information contained in the hGK-1 gene.