SUMMARY Specific antisera are used to delineate the role of macrophages, T and B cells in the expression of graft-versus-host activity. Treatment of unfractionated C57BL/6 spleen with antimacrophage serum (AMS) or anti-0 serum (A0S) decreases the graft-versus-host activity in newborn (C3H X C57)F1 mice. Treatment of spleen with anti- ß serum AßS) has no effort on the graft-versus-host activity of spleen. Density gradient centrifugation of spleen on a discontinuous bovine serum albumin gradient results in a subpopulation of cells, of medium density, which is enriched in graft-versus-host activity. The graft-versus-host reactive cells in this subpopulation are sensitive to the cytotoxic action of A0S, but no of AMS or AßS. The results suggest that the T cell is required and the macrophage can contribute to the graft-versus-host reaction. The B cells in the donor spleen are not essential for the expression of graft-versus-host reactivity. The results also suggest the existence of two subpopulations of 0-positive cells, one of light density, enriched in rosette-forming cells, and another of medium density, enriched in graft-versus-host-reactive cells.