Permanent tolerance to an experimental heterotopic cardiac allograft can be achieved by pretreatment with antilymphocyte serum (ALS) and intrathymic inoculation of donor cells. Most successful experimental protocols have employed a time lag of 2 to 3 weeks between intrathymic pretreatment and transplantation, which makes this treatment strategy impractical for clinical heart transplantation. In these experiments we modified the standard protocol by giving ALS 24 hr prior to both intrathymic injection of donor cells and heterotopic transplantation. Seven Lewis rats had intraperitoneal injection of 1 ml of ALS and 24 hr later underwent intrathymic injection of 5 X 10(7) donor Lewis-Brown Norway (LBN) splenocytes and heterotopic cardiac transplantation using an LBN donor. Mean graft survival was 24.4 days, significantly longer than the 7.8-day graft survival observed in untreated Lewis recipients (n = 5) (P < 0.02). However, graft survival was not different from that observed in Lewis rats pretreated with ALS alone (n = 5) (25.8 days, P = NS). Permanent graft survival was produced in two rats receiving only A-LS and in one rat receiving both ALS and intrathymic inoculation. In these experiments it appears that prolongation of graft survival may have been due to the effect of A-LS alone. These results suggest that there is a critical time period between intrathymic inoculation and transplantation that is needed for permanent tolerance to be induced consistently. This may be due to the kinetics of the effects of ALS on alloreactive T-lymphocytes or to a time-dependent requirement for antigen processing in the thymus.