The protease prostate-specific antigen (PSA) is a marker widely used clinically for monitoring prostatic malignancies. Under normal conditions, this enzyme is mainly involved in the post ejaculation degradation of the major human seminal protein, the seminal plasma motility inhibitor precursor/semenogelin I (SPMIP/SgI), which is the predominant protein component of human semen coagulum. PSA primary structure and activity on synthetic substrates predict a chymotrypsin-like activity whose specificity remains to be established. The present study was aimed at characterizing the proteolytic processing of the SPMIP/SgI by PSA. Purified SPMIP/SgI was incubated with PSA in the presence or absence of protease inhibitors. General serine protease inhibitors, heavy metal cations (Zn2+ and Hg2+), and the heavy metal chelator 1,10-phenanthroline partially or totally inhibited the proteolytic activity of PSA toward SPMIP/SgI. Under identical conditions, other proteins, such as bovine serum albumin, ovalbumin, and casein, were very poor substrates for PSA. Hydrolysis products were separated by reverse-phase high-performance liquid chromatography, assayed for sperm motility inhibitory activity, and analyzed by immunoblotting and mass spectrometry. The region responsible for the sperm motility inhibitory activity and containing an SPMI antiserum epitope was localized to the N-terminal portion of the molecule between residues 85 and 136. On the other hand, a monoclonal antibody against a seminal vesicle-specific antigen (MHS-5) recognized fragments derived from the central part of the SPMIP/SgI (residues 198-223). PSA hydrolysis occurred almost exclusively at either leucine or tyrosine residues, demonstrating directly for the first time a restricted chymotrypsin-like activity on a physiological substrate. The results suggest that PSA is the main enzyme responsible for the processing of SPMIP/SgI in human semen and that this protease manifests unusual specificity with respect to hydrolyzable substrates and sites of hydrolysis.