Acrosome of human sperm possesses two distinct antigens that are immunogenic, and will elicit autoantibodies that are detectable by immunofluorescence (IF). The first antigen, Acl, diffuse in distribution, is probably glycoprotein in nature since it is removed by trypsin and periodate. It is readily removed from cells after incubation in acid buffer or phosphate-buffered saline (PBS), stable at 60 degrees C and not affected by trypsin inhibitor. The second antigen, Ac2, discrete in distribution, is resistant to trypsin treatment. It remains stable after incubation in acid buffer or PBS, is unstable at 60 degrees C and becomes more diffuse in distribution when incubated in acid buffer or trypsin inhibitor. The use of spermatozoa pretreated with acid buffer permits detection of anti-Ac2 antibody that coexists with anti-Ac1 antibody in the same serum sample. Both Ac1 and Ac2 antigens are demonstrable in spermatozoa from the ejaculate, epididymis and the testis; in spermatids and spermatocytes. Ac1 antigen appears to show extensive cross-reaction with micro-organisms and with antigen(s) of human adrenal gland; and anti-Ac1 antibody is found frequently in the serum of men before vasectomy. In contrast, Ac2 antigen does not show cross-reaction with micro-organisms or tissue antigens tested; and its antibody is found mainly in the male and primarily after vasectomy. Thus, anti-Ac2 antibody may be more indicative of an immune response to sperm, and should be sought in diseases related to sperm immunity.