A sperm antigen has been isolated from radiolabeled human sperm cell membrane by detergent solubilization, lectin affinity chromatography, gel filtration, and indirect immune precipitation using sperm-immobilizing antisera from patients with unexplained infertility. Isolated material was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Among 20 infertile women's sera with sperm-immobilizing antibodies, two were found to react predominantly with a sperm membrane polypeptide having the approximate molecular weight of 15,000 daltons. No significant binding to this molecule was observed in any sera from pregnant women, unmarried women, and normal men. By the absorption with spermatozoa, the antisera lost their binding activity to the molecule, while the sera absorbed with seminal plasma did not lose the activity. The results indicated that the molecule is a genuine sperm antigen and not a sperm-coating seminal plasma antigen. By the indirect immunofluorescence of washed ejaculated spermatozoa with the antisera, strong fluorescence was localized only in an equatorial segment of the acrosome, while no specific staining was observed in the controls. The antigen is relatively unstable against acid, alkali, and heat treatment. Treatment with proteolytic enzymes such as pronase and trypsin inactivated the antigen activity, indicating that the antigen epitope could be a peptide portion of the glycoprotein.