As part of our continuous effort to elucidate the biochemical and immunological nature of human sperm surface antigens, monoclonal antibodies to human spermatozoa were generated by improved hybridoma techniques. Following immunizations with the membrane fraction of human spermatozoa and cell fusions, hybrid cells were cultured in a semi-solid HAT-selection medium to maximize the number of monoclones recovered. Subcultures were made in liquid phase 7 to 10 days after cell fusions by removing colonies from the initial medium. Based on the results of screening by microplate enzyme-linked immunoassay, 143 of 552 initial clones were found to secrete antibodies to human sperm antigens. More than one-hundred independently derived hybrid cell lines were established. Using indirect immunofluorescent procedures, 62 cell lines were shown to produce antibodies to surface antigens of human spermatozoa. Unique sperm antigens that react with monoclonal antibodies were identified by the SDS gel/protein blot radioimmunobinding method. Sperm agglutinating and immobilizing antibodies were exhibited by 4 and 15 hybrid cell lines, respectively. Fourteen of the monoclonal antibodies also exhibited cross-reactivity with methanol-fixed sperm cells of the rabbit or mouse or both whereas a reaction was not seen with viable sperm of these species. Generation of monoclonal antibodies against a wide spectrum of human sperm antigens should facilitate future investigations regarding immunologic-associated human infertility and fertility control.