Seminal plasma, an amorphous material that exists in semen, contains proteins related to sperm forward motility. Employing affinity chromatography with ConA beads and protein ultrafiltration, we isolated and concentrated proteins from heated human seminal plasma. Results of computer-assisted semen analyses (CASA) demonstrated that the forward motility index of bovine spermatozoa from the epididymal caput, incubated with proteins and theophylline, was significantly different from that of spermatozoa incubated with theophylline alone (P < 0.01). The electrophoreses revealed that the protein bands with high molecular weights in the gel of PAGE changed into low molecular weights in the gel of SDS-PAGE. Furthermore, proteins from a separated portion of the PAGE gel were still able to stimulate spermatozoa from the epididymal caput to gain forward motility. Two-dimensional (2D)-gel electrophoresis and mass spectrometry indicated that spots focused on the portion seemed, according to their amino acid sequences, to be like human alpha-1-antitrypsin and zinc-alpha-2-glycoprotein (ZAG) precursors. Western blot analysis showed the presence of these two proteins in seminal plasma. These proteins, related to the forward motility of spermatozoa in human seminal plasma, may play important roles during maturation of spermatozoa, from the epididymis through fertilization in the female reproductive tract.