The in vivo significance of suppressor macrophages for antitumor immunity was investigated in a syngeneic tumor system. The presence of suppressor macrophages in the spleens of X5563 C3H/HeN tumor-bearing mice (TBM) was directly shown in vitro. Thus the addition of splenic macrophages of TBM suppressed the in vitro secondary induction of both tumor-specific cytotoxic T-cells and effector cells of the delayed-type hypersensitivity reaction. Splenic macrophages of TBM exerted suppression on tumor-specific T-cell-mediated cytotoxicity in the effector phase as well. Prostaglandin production was found to be one of the major mechanisms involved in macrophage-induced suppression. In vivo treatment of TBM with carrageenan and/or indomethacin retarded tumor growth and in parallel augmented cell-mediated cytotoxicity against X5563 cells, probably by affecting suppressor macrophages in vivo. The suppressive effect of splenic macrophages from TBM was clearly demonstrated in a tumor-neutralization test indicating that suppressor macrophages were able to exert their function in vivo as well as in vitro. All these results suggested that suppressor macrophages had in vivo significance for the suppression of the immunosurveillance of the hosts.