Experiments were performed to determine whether antibody-producing hybridomas could be derived by fusion of BALB/c P3X63Ag8 myeloma cells and spleen cells from C57BL/6 mice either immune to or bearing a syngeneic methylcholanthrene (MCA)-induced fibrosarcoma (M3). Mice were immunized by tumor amputation followed by injections of tumor cells, purified tumor cell membranes, or murine leukemia virus isolated from M3 cells (M3-MuLV). Tumor-bearer spleens were obtained at weekly intervals over a 6-week course of tumor bearing. Hybridoma cultures showing the best growth in selective HAT medium were screened by radioimmunoassay for antibody production against a) viable and glutaraldehyde-fixed cultured M3 cells, b) glutaraldehyde-fixed in vivo-passaged M3 cells, c) purified M3 cell membranes, and d) M3-MuLV. Stable antibody-producing cloned hybridomas were obtained only from fusions involving tumor-bearer spleen cells. One clone (2C2-A1) secretes an IgM antibody that binds well to purified M3-MuLV and M3 membranes, but only weakly to intact M3 cells. Another cloned line (3A5-L1) secretes an IgG1 antibody that binds to M3 cells but not to M3-MuLV. Further analysis of specificity by binding and absorption studies involving more than 30 other normal and transformed murine cell types indicates that 3A5-L1 antibody binds to some, but not all, MCA-induced murine fibrosarcomas. 3A5-L1 antibody demonstrates little, if any, binding to murine tumors of other histologic types or to normal tissues. The results demonstrate that it is possible to capture and perpetuate part of the tumor-bearing host's antibody repertoire in the form of hybridomas secreting monoclonal antibodies specific for tumor-associated antigens.