An acute graft-vs.-host reaction (GVHR) was induced by i.v. injection of 10(8) lymphoid cells from C57BL/10 (B10) donors (H-2b/b) into adult non-irradiated (B10 X DBA/2)F1 mice (H-2b/d). Previous experiments have established that spleen cells obtained from such GVHF1 mice suppress the primary antibody response of normal F1 spleen cells to sheep red blood cells. This type of suppression was termed "allosuppression", and it was shown to be mediated by Ly-2+ T cells of donor origin that react against H-2 antigens of the host. It was unclear, however, whether the actual mechanism of allosuppression was due to suppressive factors generated by donor T cells or whether the latter killed the F1 B cells. Here, we show that for their suppressive effect GVHF1 spleen cells need direct cell contact with F1 spleen cells; no suppressive effect was measured in a double-chamber culture system in which the GVHF1 spleen cells were separated from the responding normal F1 spleen cells by a cell-tight membrane filter. The suppressive effect only affected cells expressing the appropriate H-2 class I alloantigen; in mixed cultures of irradiated F1 spleen cells and GVHF1 spleen cells with third-party B cells the antibody response of the third-party B cells was not suppressed. GVHF1 spleen cells showed cytotoxic T lymphocyte (CTL) activity specific for the allogeneic F1 H-2 antigen. The suppressive effect of the GVHF1 spleen cells did not differ from that exerted by cloned CTL specific for MHC class I alloantigens; cloned CTL suppressed the primary antibody response of murine spleen cells without affecting the response of third-party B cells added to the cultures. The combined findings show that "allosuppression" in vitro is not due to suppression of F1B cells, but to a direct killing of these cells by alloreactive CTL.