Hybridoma cell lines were produced by PEG (polyethylene glycol)-induced fusion of myeloma cells (NS-1/X63 Ag 8) with dispersed spleen cells from a Balb/c male mouse immunized syngeneically with homogenized Balb/c testis. Indirect immunofluorescence on mature (cauda epididymal) mouse sperm was employed as the primary screening assay. This procedure allowed the immediate identification of hybridomas which produce monoclonal antibodies of interest with respect to the fertilization process. One hybridoma cell line, M29, secretes an antibody of the IgM class that localizes to the equatorial segment of the mouse sperm head. This antibody is not species specific, but is restricted to the homologous functional area, the equatorial segment, in all other sperm examined (hamster, rabbit, human). When tested for a possible effect on the fertilization process, ascites fluid containing M29 antibody was very effective in blocking the fertilization of mouse gametes in vitro. The same inhibiting activity was observed regardless of whether the M29-treated sperm first encountered the cumulus cell layer, the zona pellucida, or the egg plasma membrane. A large number of M29-treated sperm were capable of penetrating zonae pellucidae when this layer was present. It appears, therefore, that M29 monoclonal antibody prevents fertilization specifically at the level of interaction between the sperm and egg plasma membranes; the other prefertilization events (hyperactivated motility, zona binding, acrosome reaction, zona penetration) seem little affected. Using the M29 antibody and related probes that are specific for particular biological events, it should be possible to map, in molecular terms, the functional domains of the sperm cell's membrane.