Cocultivation of spleen cells, lymph node cells, and thymocytes of female Wistar-King-Aptekman rats with short-term cultured male adult T cell leukemia (ATL) cells in the presence of 5-bromo-2'-deoxyuridine (BrdUrd) resulted in the establishment of rat lymphoid cell lines, TARS-1, TARL-2, and TART-1. Cytogenetic analysis of the three cell lines showed a female rat karyotype with 42 chromosomes. The surface phenotypes of TARS-1 and TART-1 were those of rat T cells. TARL-2 was only positive for rat Ia and leukocyte common antigens. The cell lines continuously produced a type C retrovirus, human T cell leukemia virus (HTLV) and expressed ATL-associated antigens. TARS-1 and TART-1, but not TARL-2 were transplantable into newborn syngeneic rats and nude mice. These results strongly indicate that HTLV not only immortalizes, but also transforms rat T cells in vitro. Adult rats immunized with either TARS-1 or TARL-2 produced antibodies specific for HTLV. The biochemical analysis of the antigens that reacted with rat sera revealed that they are the two HTLV-specific polypeptides, p24 and p28.