Tumor progression, lung metastasis, and death occur in tumor-bearing BD IX syngeneic rats in a fashion similar to the course of patients with metastatic colon cancer. In an effort to establish a relevant model for monoclonal antibody (MoAb) therapy of tumors, we generated murine MoAb against DHD/TR, a dimethylhydrazine-induced rat colon carcinoma which has been adapted to cell culture. Murine MoAb 17B10 E4 (E4) reacts with the TR tumor and shows weak immunoperoxidase reactivity with normal rat tissues. Murine MoAb 5F7 D3 (D3) reacts with the tumor and a variety of normal rat epithelia. Both are IgG2a and mediate cytotoxicity by rat peripheral blood mononuclear cells. 18D5 F6 (F6) also reacts with the tumor and normal tissues but is an IgG2b and does not mediate cytotoxicity in the presence of rat effector cells. Iodinated E4 and D3 antibodies retained their immunoreactivity. E4 revealed 9.8 × 105 antigenic sites per TR cell, with an affinity constant of 9.35 × 107 m-1, while D3 demonstrated 2.5 × 106 antigenic sites and an affinity constant of 4.2 × 107 m-1. Immunoblotting showed that the antigens recognized by D3 and E4 are glycoproteins with molecular weights of 27,000 and 66,000, respectively. F6 failed to react with its antigen present in the blot. This rat colon carcinoma and the monoclonal antibodies described here may provide experimental data useful for implementing monoclonal antibodies in cancer therapy.