Growth of the secondary Meth 1 tumors, which had been inoculated s.c. in the abdomens of BALB/c mice bearing primary Meth 1 tumors in the flanks, was inhibited as compared with that of tumors in the normal mice, suggesting the development of concomitant antitumor immunity. When levamisole (LMS, 0.625 or 2.5 mg/kg) was administered i.p. to the Meth 1-bearing mice daily before or after secondary inoculation, growth inhibition of secondary tumors was augmented. This effect of LMS was suggested to be tumor specific as evidenced by no growth inhibition of secondary Meth A tumors in mice bearing primary Meth 1 tumors. The spleen cells of 11-day Meth 1-bearing mice exhibited growth-inhibitory activity against Meth 1 cells in the Winn assay. Administration of LMS augmented the growth-inhibitory activity. This effect of LMS was mediated by nonadherent spleen cells and completely lost by the preincubation with anti-Thy 1.2 antibody and complement. Cytotoxicity was detected in the spleen cells of Meth 1-bearing mice by 51Cr release assay after in vitro sensitization with mitomycin C-treated Meth 1 cells. In vivo administration of LMS augmented the cytotoxicity, which was tumor specific and completely lost by the preincubation of the spleen cells with anti-Thy 1.2 antibody and complement. These results suggest that the growth-inhibitory effect of LMS against secondary tumors was mediated by cytotoxic T-cells.