Immunoglobulin G (IgG) fraction was prepared from a serum obtained from an infertile woman containing antisperm antibodies that induced head-to-head agglutination of human sperm. The antibodies in the IgG fraction interacted with a 60-kD protein found in human testes determined by Western blot. The 60-kD protein was purified from human testis by isoelectric focusing (IEF), affinity chromatography on blue sepharose column, and preparative electrophoresis with electroelution. The purified 60-kD protein migrated as a single homogeneous band when analyzed by SDS-PAGE. The amino acid sequence of the N-terminus was determined. The initial 10 amino acid residues were identical to the human serum vitamin D binding protein (VDBP). Polyclonal antibodies were raised against the 60-kD protein. The polyclonal anti-60-kD antibodies and the anti-VDBP antibodies obtained from a commercial source immobilized human sperm in vitro. The interacting antigens were located on the postacrosomal region and midpiece of human sperm, as determined by an immunofluorescence method. The IgG fraction prepared from the serum of an infertile woman interacted with the human testis 60-kD protein but failed to stain serum VDBP. The results suggest that the 60-kD and VDBP are related proteins but not identical entities and that the 60-kD protein contains a unique structural group lacking in serum VDBP. Production of antibodies against the unique structure of the 60-kD protein may be the cause of the infertility.