Tumor-associated transplantation antigen (TATA) was solubilized with n-butanol from rat gliosarcoma cells (T-9). Under the conditions used for the solubilization of TATA, the viability of T-9 cells was maintained above 85%, suggesting that the contamination of TATA with cytoplasmic protein is minimal. Normal Fischer rats were treated with 500 micrograms of n-butanol extract and inoculated with 2 X 10(6) viable T-9 cells. At 8 weeks after the inoculation of viable tumor cells, the tumor diameter was significantly smaller than that in control untreated rats. Rats treated with 770 micrograms of n-butanol extract from T-9 cells were inoculated with 1 X 10(7) syngeneic FTL-13 thymic lymphoma cells or T-9 cells. The rats rejected the T-9 cells, whereas the FTL-13 cells grew gradually until the host died, indicating that the n-butanol extract from T-9 cells contained T-9 cell-specific TATA. The n-butanol extract was characterized by chromatographic separation on a TSK gel G3000SW gel filtration column and a Mono Q anion exchange column with the fast protein liquid chromatography system (FPLC). The TATA of T-9 cells was found to have a molecular weight of approximately 40,000-70,000 and was eluted at 0.6-0.9M NaCl from the Mono Q column. The cellular mechanisms responsible for the rejection of T-9 cells in the rats treated with the n-butanol extract were also examined. Adoptive transfer and in vitro experiments demonstrated that helper T cells for the generation of cytotoxic T lymphocytes were generated in the rats treated with n-butanol extract.