Seven stable hybrid cell lines producing monoclonal IgG antibodies reacting with H-2 and Ia antigens have been established. The lines were obtained by fusing NS-1, a nonsecreting variant of MOPC-21 (Köhler, G., Howe, S. C., and Milstein, C., Eur. J. Immunol. 1976. 6: 292) with spleen cells from either BALB/c or C3H.SW.Igb (CWB) mice that had been immunized with H-2k haplotype spleen cells. One of the monoclonal antibodies reacts with H-2Kk determinants; the remaining six react with determinants mapping to the I-Ak subregion. The antigens bound by the anti-I-Ak antibodies were analyzed by two-dimensional electrophoresis and were found to consist of the same polypeptide chains as are precipitated by anti-I-Ak alloantisera. The Ia antigenic determinants recognized by these monoclonal antibodies and their presence on lymphocyte subpopulations have been characterized by 125I-binding assays, complement-mediated cytotoxicity, and immunofluorescence, by using the fluorescence-activated cell sorter (FACS).