SummaryFemale mice of the BALB/c isogenic strain were immunized with epididymal sperm of the male mice. Four antigenic perparations were used: whole sperm (WS), homogenized sperm (HS), protein fraction (PF) and lipid fraction (LF). Antisera were assayed for antibody activity by sperm immobilization and indirect immunofluorescence tests. The differential results between the WS and HS antisera obtained in the two tests suggest that sperm membrane surface antigen(s) may be distinct from intracellular antigen(s). PF‐antiserum showed lower but significant activity with the sperm immobilization test but weak activity with the indirect immunofluorescence test. The lipid fractions showed extremely weak activities with both tests. Indirect immunofluorescent staining was curried out on 10 μm sections of unfertilized and fertilized eggs and 2‐cell embryos. Unfertilized eggs showed no antibody binding and fertilized eggs showed patchy fluorescence of the plasma membrane and cytoplasm. In the 2‐cell embryo the fluorescence was diffuse, suggesting spreading of the sperm antigens. Comparative electrophoresis of aliquots of HS antiserum with and without incubation with washed sperm showed a clear reduction, due to adsorption by sperm, of IgG class of antibodies, on the basis of molecular weight. The differences in antigenicity between the four sperm preparations are discussed in relation to (i) possible localization of the antigens in the spermatozoa and (ii) the conformational differences of the antigens manifested by organic solvent denaturation.