Adenovirus type 12 (Ad12)-transformed mouse cells were examined for their susceptibility to cell-mediated immunity in vitro, with respect to the activity of the virus-specific surface (S) antigen in the cells. A transformed cell line, C57ATl, was established from embryonic cells of C57BL/6 mice by Ad12 infection. In fluorescent antibody tests, the transformed cells were positive for the S antigen when the cells were maintained as cultures, whereas when the cells were grown as tumors in animals they became negative for the antigen (referred to as S(+) and S(-) cells, respectively). These S(+) and S(-) cells were subjected to the 51Cr-release test for cell lysis by immune spleen cells (ISC) raised in syngeneic mice by Ad12 infection. When the S (+) cells at various passage levels were exposed to ISC, all of them were lysed extensively and to a similar extent irrespective of their passage history. In contrast, the S(-) cells were consistently refractory to the action of ISC. In addition, the cytotoxic action of ISC was markedly impeded by pretreating the S(+) cells with antiserum to the S antigen, or the ISC with anti-Thy-1,2 serum plus complement. Taken these findings together, the S(+) cells were assumed to be injured by ISC through direct interaction of the S antigen with T-lymphocytes.In the tumors induced by adenovirus type 12 (Ad12), a virus-specific antigen has been located in their cell surfaces by the in vitro methods of cytotoxicity (2, 5, 17, 27, 28) and of fluorescent antibody (FA) technique (15,21,31), and the in vivo method of immunorejection revealed a virus-specific TSTA (tumor-specific transplantation antigen) (3-7, 11, 12, 18, 22-24, 28-30). However the relationship between the two antigens is not yet fully established. Of these antigens, the one shown by the FA technique and called surface (S) antigen was readily detectable in Ad 12-transformed cells cultured in vitro (15, 21), but not in tumor cells grown in animals unless the cells were treated with a low concentration of trypsin (31). In a previous paper, we described alternate changes of the S antigen on Ad12-transformed cells cultured in vitro and grown in animals (21). In the FA test, while all of the hamster and mouse cells transformed with Ad12 showed specific and distinct membrane fluorescence, the tumor cells produced by grafting the transformed cells, did not show any fluorescence (referred to as S(+) and S(-) cells, respectively). In the same paper, it was also shown that the S(+) cells were less tumorigenic and more immuno-1085