We have previously demonstrated in a rat renal allograft model, using adoptive transfer studies, that a single blood transfusion results in the generation of blood donor-specific W3/25+ (CD4) T suppressor cells detectable in the thoracic duct lymph, but not in the spleen, 7 days after transfusion. In this study, again using the LEW-to-DA strain combination, we have investigated the time of appearance of suppressor activity in different lymphoid compartments in vivo and in vitro following a single blood transfusion. Cells (5 x 10(7) unfractionated TDL or 1 x 10(8) unfractionated spleen cells) were harvested from DA rats on days 1-6 after a single LEW blood transfusion and were adoptively transferred into syngeneic (DA), lightly irradiated (200 rads) hosts. The following day these animals received a kidney allograft (LEW or PVG). Specific suppressor activity, as demonstrated by the prolongation of survival of LEW, but not PVG, renal allografts was noted in the spleen 4 days after transfusion (MST greater than 100 days). Suppressor cells were not found in the TDL at this stage--but 6 days after transfusion suppressor activity could be detected in the TDL (median survival time [MST]greater than 100 days), while at this time no such activity was demonstrable in the spleen. The suppressor activity in the spleen on day 4 after transfusion was shown to be mediated by a W3/25+ (CD4) T cell. The proliferative response in a mixed lymphocyte culture of DA spleen cells, prepared 4 days after transfusion, or TDL cells prepared 6 days after transfusion, to LEW stimulator cells was significantly suppressed when compared with the response of cells harvested from untreated controls. In contrast, the proliferative response of spleen cells examined 6 days after transfusion, when no suppressor activity was detected in vivo, was significantly augmented in comparison with untreated controls. Thus we have demonstrated that a DST results in the generation of W3/25-positive (CD4) T cells capable of suppressing renal allograft rejection that appear transiently in the splenic compartment (4 days after transfusion) and subsequently in the TDL (six days after transfusion).