Luminal contents of guinea pig seminal vesicle were extracted with 0.154 M NaCl and the soluble protein fraction was studied by DEAE-cellulose and Sephadex G-100 column chromatography; anionic, cationic, and SDS-polyarcylamide disc gel electrophoresis; isoelectric focusing; molecular weight determinations by SDS-polyacrylamide disc gel electrophoresis; analysis for neutral carbohydrate; sucrose density-gradient centrifugation for determination of sedimentation coefficients; and amino acid analyses. Four protein fractions (referred to as proteins 1, 2, 3, and 4 in order to elution) were obtained by DEAE-cellulose column chromatography. Proteins 1, 2, and 4 gave single major bands in multiple disc gel electrophoretic systems. Protein 3 gave one major band and one minor component believed to be a fragment of a primary vesicular protein. Protein 1 is very basic and probably represents the clotting protein of guinea pig semen. The three nonclotting proteins could not be detected in serum and are probably intrinsic to seminal vesicle epithelium. All four proteins were found in animals weighing 700 g or more; only two of the three nonclotting proteins were found in more than half of the younger animals. This makes it even more likely that one or more of the nonclotting proteinc could serve as a useful gene marker in studies of androgen mechanisms.