The cellular requirements for rejection of heart grafts bearing isolated major histocompatibility complex (MHC) subregion RT1A-encoded class I disparities was assessed by adoptive transfer. Sublethally irradiated (780 rads) (PVG X WF)F1 recipients of irradiated PVG-RT1r1 heart grafts were selectively reconstituted with spleen cells from syngeneic donors previously sensitized with two sequential PVG-RT1r1 skin grafts. PVG-RT1r1 heart grafts were rejected acutely in recipients reconstituted with 10 X 10(6) unfractionated immune spleen cells (7-9 days, n = 6) or inocula (5 X 10(6) cells) depleted of SIg+ cells (10-13 days, n = 5), but additional depletion of cytotoxic T cells and their precursors (OX8+) resulted in marked prolongation of graft survival (n = 4, 4 X 10(6) cells, 45-67 days). Reducing the reconstituting inocula from 4 X 10(6) to 2.5 X 10(6) spleen cells (OX8-, SIg-) prolonged graft survival to that observed in unreconstituted recipients (generally greater than 100 days). Additional studies were performed to define the immunologic basis for prolonged survival of PVG-RT1r1 heart grafts in homozygous PVG recipients. Although lymphoid cells of naive PVG failed to proliferate (stimulation index [SI] 1.01, P = NS) on coculture with irradiated PVG-RT1r1, bulk cultures yielding but weak and variable CTL generation, lymphoid cells from specifically sensitized PVG proliferated (SI 4.25, P less than .001) and generated greater cytotoxic T lymphocyte (CTL) activity (P less than .001) under identical conditions, strongly suggesting, therefore, that prolonged heart graft survival in this strain combination is related to low CTL precursor frequency. Further, though PVG-RT1r1 heart grafts were rejected in 10-12 days (n = 3) by (PVG X WF)F1 recipients, (PVG-RT1r1 X WF)F1----(PVG X WF)F1 heart grafts (RT1Aa disparity) survived greater than 100 days despite an equivalent alloimmune response, and this was shown to correlate with a reduced sensitivity of (PVG-RT1r1 X WF)F1 target cells to lysis by CTL. These data, therefore, strongly suggest that the pivotal role of CTL in the rejection of class-I-disparate heart grafts is, in fact, related to their function in direct cell-mediated cytolysis.