Inhibition of human sperm hyperactivation by an anti-azido sperm monoclonal antibody. After a period of hyperactivation in vitro, the majority of human spermatozoa become immotile and become susceptible to induction of acrosome reaction by progesterone. Therefore the mechanism of the hyperactivation process was investigated. We have shown that sperm membrane gangliosides such as GM1, GD1b, GT1b, and GQ1b stimulated hyperactivation of spermatozoa from fertile donors and from infertile patients with and without immotile sperm disease. In these experiments, a purified anti-sperm monoclonal antibody with IgG subclass lambda recognized a single band with apparent m.w. greater than 200,000 in extracts of sperm cells. This protein appears to be in the membrane of spermatozoa and was expressed on the flagellum of hyperactivated sperm after treatment with the compound Bt2cAMP and was localized to the sperm midpiece and tail. A purified anti-azido sperm monoclonal antibody with IgG subclass lambda recognized bands of m.w. 98,000, 64,000, 52,000, 43,000, and 28,000 in this same extract of human sperm. This antibody inhibited human sperm hyperactivation in vitro by blocking motility, the acrosome reaction, and capacitation. A rabbit antiserum produced against this monoclonal antibody showed a similar effect to that of the monoclonal antibody. These results indicate that one of the roles of a sperm cell membrane protein or proteins with a molecular weight between 43,000 and 28,000 is to stimulate the hyperactivation of spermatozoa from infertile patients and fertile donors. This protein or proteins appears to be linked to the plasma membrane of spermatozoa. The monoclonal antibody and antiserum appear to inhibit normal sperm functions through binding to these same proteins. The significance of the interaction between these sperm surface proteins and the monoclonal antibody and antiserum will be elucidated by developing antibodies to these proteins.