We have produced lymphocyte hybridomas between mouse myeloma cells and either spleen cells of C3H/f/C57BL/6 mice bearing the Mm5mt/c1 tumor-producing murine mammary tumor virus (MMTV) or spleen cells from Fisher rats inoculated with the same tumor. Two classes of hybridoma-secreted monoclonal antibodies were obtained. In the first class are IVC11, IIIA1, and VE7, each of which precipitated a 52,000-dalton protein from 125I-labeled purified preparations of MMTV and [3H]glucosamine-labeled Mm5mt/c1 cell extracts. A second class of monoclonal antibodies, represented by IC12 and IIIB5, reacted specifically with C3H MMTV-secreting cells in radioimmunoassays (RIA) with whole cells and did not precipitate proteins with labeled virus or metabolically labeled Mm5mt/c cell extracts. IC12 and IIB5 could be distinguished from each other in a RIA in which antigen discs were prepared from a membrane preparation of Mm5mt/c1 cells. In this assay, IC12 antibody was blocked from reacting with discs precoated with goat anti-MMTV, and IIB5 antibody was not.