We have recently found that donor-specific tolerance to a cardiac allograft can be achieved after the intrathymic (i.t.) injection of donor splenocytes and a single intraperitoneal injection of rabbit antirat lymphocyte serum. The present study evaluated whether the tolerance induced by splenocytes injected i.t. could also prevent the rejection of kidney and skin allografts. Male Buffalo (RT1b) rats were given 25 x 10(6) fully MHC-mismatched unfractionated Lewis (RT1l) splenocytes by i.t. injection plus 1 ml of ALS i.p. and 21 days later underwent a Lewis heterotopic cardiac, orthotopic renal, or skin transplant. Lewis i.t. injection induced a donor-specific tolerance with indefinite cardiac allograft survival (> 153.1 days) in 88% of the recipients without the need for further immunosuppression, while renal and skin allograft survival was prolonged (kidney 14.8 days vs. control 7.8 days; skin 11.6 days vs. control 9.2 days) but were still rejected. Buffalo recipients with a long-surviving Lewis cardiac allograft after Lewis i.t. injection were still able to reject a third-party heterotopic ACI (RT1a) cardiac allograft in normal time (7.0 days), but did not reject a second Lewis cardiac allograft (> 100.0 days). In contrast, however, Buffalo recipients with long-surviving Lewis cardiac allografts did reject a Lewis skin allograft in normal time (10.0 days) and a Lewis renal allograft in a prolonged manner (17.6 days) without causing the rejection of the Lewis cardiac allografts. These data support the important role tissue-specific non-MHC antigens may play in the rejection of kidney and skin allografts.