Mouse hybridomas were isolated that produce monoclonal antibodies (MAbs) to several regions of the HIV envelope, gp120, which may be used to map immunogenic regions in animal and human immunity. One series of MAbs was generated by repeated immunization with recombinant gp120 (rgp120) from a single isolate, and a second series by sequential immunization with rgp120 from three isolates. All MAbs bound to rgp120IIIB, but only one bound well to cell surface-expressed gp160. Synthetic peptides spanning much of the length of gp120 were used to map MAb reactivity. Two MAbs were mapped to the C1 region, one to the C2 region, and three to the C5 region by this approach. Six distinct epitopes were detected by competitive binding analysis. One MAb binding the C1 region blocks binding of CD4 to rgp120IIIB, binds by ELISA to rgp120MN (an isolate not used during immunization), and binds to cell surface-expressed gp160. These hybridomas will be made available to the scientific community through a hybridoma culture facility.