Suppressor cells from syngeneic P815 mastocytoma-bearing DBA/2 mice that inhibit in vitro generation of specific anti-tumor cytotoxicity were characterized. Suppressive activity was almost completely eliminated by treating suppressive spleen cells with anti-θ serum and complement. Treatment with anti-mouse Ig serum and complement or with carbonyl iron did not affect their suppressive activity. When suppressive thymocytes from P815 tumor-bearing DBA/2 mice were tested for their capacity to inhibit the generation of cytotoxicity against L1210 cells, a leukemia line in DBA/2 mice, they did not affect the activity, indicating that the suppressor cells in the thymocytes of P815 tumor-bearing mice are specific to the tumor. When Ficoll-Hypaque density cell separation was carried out with cytotoxic spleen cells and suppressive spleen cells from P815 tumor-bearing mice, the dense fraction was enriched for killer cells whereas the suppressive activity was mainly recovered in the light fraction. Therefore, killer cells and suppressor cells in P815 tumor-bearing mice are thought to be distinct populations.