An L3T4-, Lyt2+ tumor-specific, cloned T-lymphocyte cell line (RTT-2) was isolated from a spleen cell population harvested from C3H/HeJ mice, following in vivo immunization against a syngeneic MCA-induced fibrosarcoma (MCA-F) and in vitro restimulation with 1-butanol-extracted, isoelectrophoretically purified MCA-F tumor-specific transplantation antigen (TSTA). RTT-2 required exposure to homotypic-extracted MCA-F TSTA in combination with low-dose IL-2 to maintain its specific cytotoxic activity in vitro. In vivo local adoptive transfer of RTT-2 caused specific neutralization of homotypic MCA-F, but not heterotypic MCA-D, tumor cells. Systemic in vivo transfer of RTT-2 alone augmented host resistance. In combination with a triple regimen of weekly doses of purified TSTA (1 microgram SC) and a single ip injection of CY (20 mg/kg), adoptive transfer of RTT-2 cells (1 X 10(7)) retarded the neoplastic outgrowth in and prolonged the survival of primary hosts bearing 3-, 7-, and 14-day established MCA-F tumors. In a spontaneous pulmonary metastasis model following amputation of a tumor-bearing limb, the triple regimen of TSTA/CY/RTT-2 markedly reduced the number of lung colonies. Thus RTT-2, which displays specific tumoricidal activity in vitro and in vivo, may afford a suitable tool to dissect T-cell receptors recognizing tumor markers on 1-butanol-extracted, MCA-F TSTA.