ABSTRACT: Female mice were isoimmunized with homologous spermatozoa of the same strain. Hybrid cells that secrete monoclonal antibodies to mouse sperm isoantigens were generated by modified hybridoma techniques using a semi-solid Iscove's modified Dulbecco's medium containing methylcellulose for the initial cloning. Out of more than 1,000 colonies that were initially recovered for subculture, 246 were shown to produce antibodies reacting with various cytological regions of mouse spermatozoa, when methanol-fixed sperm were employed in an indirect immunofluorescent assay. More than 75% of the generated monoclonal isoantibodies were shown to bind the acrosomal regions of mouse spermatozoa. Some were found to cross-react with spermatozoa from other mammalian species including those of human, rabbit, rat, and guinea pig. However, none were shown to cross-react with mouse lymphocytes. Two-thirds of the generated monoclonal antibodies can also bind live mouse spermatozoa. By an immunohistochemical technique using testicular sections, some of these monoclonal antibodies were shown to react with specific antigens expressed during different stages of spermatogenesis. It is concluded that these mouse sperm isoantigens are sperm-specific and appear uniquely during spermatogenesis. Monoclonal isoantibodies produced in the present study may have potential applications regarding the investigations of sperm iso- or autoimmunity, spermatogenesis, and fertility control.