Mutagen treatment of mouse mastocytoma P815 produces highly immunogenic "tum-" variants. Most of these variants express potent transplantation antigens which are not present on the original P815 tumor cells. These tum- antigens, which appear to be specific for each variant, elicit a strong cytolytic T lymphocyte (CTL) response, but do not seem to induce a specific antibody response. As a first step in the isolation of the gene of a tum- antigen, we attempted DNA-mediated gene transfer. As a DNA recipient cell we used P1.HTR, a highly transfectable P815 cell line, whose selection has been previously described. For the detection of antigen-expressing cells in transfected populations we developed a procedure that relies on the ability of these cells to stimulate the proliferation of the relevant CTL. Using DNA from tum- variant P91 mixed with a plasmid carrying an antibiotic resistance gene, we obtained several independent transfectants expressing a tum- antigen, at a frequency of approximately 1 in 13,000 antibiotic-resistant transfectants. These transfectants express only one of the two tum- antigens that were identified on P91, suggesting that these tum- antigens correspond to different genes. We expect that the detection procedure described here will be suitable for the identification of transfectants for any gene that determines the expression of an antigen recognized by CTL.