The proteins in human seminal plasma were mapped by high-resolution two-dimensional electrophoresis (ISO-DALT and BASO-DALT systems). When analyzed under dissociating conditions, samples from normal fertile males revealed a pattern of over 200 proteins, ranging in mass from 10 000 to 100 000 daltons. Comparison of the mapped proteins from these males and those who had undergone vasectomy allowed us to identify one series of glycoproteins as missing from the semen from vasectomized individuals. Glycoproteins isolated by affinity chromatography with use of concanavalin A were also mapped. Some of the protein spots were identified either by co-electrophoresis with purified proteins or by the electrophoretic transfer of proteins to nitrocellulose sheets and subsequent detection by immunological procedures. The proteins identified include a number of serum proteins as well as prostatic acid phosphatase and creatine kinase. Proteolytic events shown to occur during the liquefaction of semen that occurs early after collection indicate the importance of carefully controlled collection and preparation methods for clinical evaluation of seminal plasma. Ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride inhibit this proteolysis.