After an i.p. transplantation of an allogeneic tumor (Meth A) to C57BL/6 mice, a macrophage (M phi)-rich, non-T, non-NK cell population is induced as the major infiltrate and cytotoxic cells. We here evaluated the role of the M phi s in the rejection of allografted Meth A cells and characterized the M phi s in comparison with other well-known M phi s. At all time intervals after transplantation, the highest cytotoxic activities against Meth A tumor were obtained with the M phi-rich population. In addition, the lymphocyte-rich population had a significant but low cytotoxic activity, whereas two other population types, granulocytes and large granular cells, were inactive. When the M phi-rich or the T cell-depleted M phi-rich population was i.p. transplanted simultaneously with Meth A cells into untreated C57BL/6 mice, the tumor cells were rejected without growth. After specific elimination of M phi s by in vivo application of dichloromethylene diphosphonate-containing liposomes, the cytotoxic activity against Meth A cells was hardly induced at the transplantation site of Meth A cells and the allografted Meth A tumor continued to grow, indicating that a type of M phi is the effector cell essential for the rejection. In contrast to other well-known M phi s, the cytotoxic activity against Meth A cells was cell-to-cell contact dependent and soluble factor (e.g., NO and TNF-alpha) independent. Moreover, the cytotoxic activity of the M phi s (H-2b) against 51Cr-labeled Meth A (H-2d) cells was inhibited by the addition of unlabeled H-2d, but not H-2b, H-2k or H-2h, lymphoblasts as well as Meth A cells, implying the specific interaction of the M phi s with H-2d cells.