The cytolytic T lymphocyte (CTL) response of syngeneic mice to antigenic variants obtained by mutagenesis of mastocytoma P815 was analyzed at the clonal level. Estimates of the frequency of CTL precursor cells in spleens from mice immunized with P815 variants ranged from 10(-4) to 2 X 10(-3). This frequency could be increased approximately 100 times by stimulating immune spleen cells in mass culture for 6-8 days with the immunizing variant. Specificity analysis of a large number of individual CTL clones demonstrated the existence of two distinct populations of CTL. Some CTL clones lysed exclusively the immunizing variant, while others lysed equally well all P815 targets, but not syngeneic tumor L1210. These results provide direct evidence for the existence of new, individual specificities on P815 variants in addition to a common antigen already present on the original P815 cells. This confirms that a variety of new antigens can be induced by mutagenesis on the same background cell. Stable, highly active CTL clones specific for either individual variant antigens or common P815 antigens could be maintained in long-term culture.