There is now convincing evidence for the imposition of self tolerance by means of the clonal deletion of self-reactive T cells operating within the thymus. Since not all self components may be encountered there, the question must be asked whether tolerance can occur post-thymically. To test this, we and other investigators have used transgenic technology to direct expression of a known "nonself" gene to a given extrathymic tissue. No lymphocytic infiltration was ever seen in transgene-expressing tissues, even if the mice were given normal syngeneic (nontransgenic) spleen cells intravenously or were stimulated with H-2Kb spleen cells. Infiltration did, however, occur in irradiated transgenic recipients of H-2Kb immune spleen cells. In MET-Kb mice, this infiltrate diminished with time, raising the possibility that peripheral tolerance may even have been induced in immune cells. H-2Kb-bearing skin was accepted in young RIP-Kb mice but rejected in older mice, which had lost more than 75% of their beta cells as a result of the overexpression of H-2Kb. This loss of tolerance thus occurred when the concentration of the tolerogen, H-2Kb, fell below some critical threshold. Following in vitro stimulation, spleen cells from young RIP-Kb mice could not kill H-2Kb-bearing targets, but could respond to third party targets. Thymus cells, on the other hand, could be stimulated to kill both targets, clearly indicating that tolerance was not imposed intrathymically. Spleen cells from older RIP-Kb mice (those that had lost most of their beta cells) killed both targets, which is in agreement with the in vivo data. Reactivity to H-2Kb was restored to young spleen cells by providing them with IL-2. Two hypotheses were proposed to account for the above findings: tolerance results either from the deletion or functional silencing of high-affinity effector cells or of regulatory, IL-2-producing helper T cells. As it is difficult to distinguish between these, we have produced a second series of transgenic mice (F3+) with rearranged TCR genes encoding an anti-H-2Kb TCR and derived "double-transgenic" (F3+RIP+) offspring by mating these mice with RIP-Kb mice. The transgenic TCR utilized the V beta 11 segment which can be detected by a monoclonal antibody. There were in the thymus very few CD4+ and very few CD4+8+ cells in both F3+ and F3+RIP+ mice and, in the double-transgenic mice, there was no evidence of deletion of CD8+V beta 11+ cells in the periphery although they showed tolerance to H-2Kb-bearing skin.(ABSTRACT TRUNCATED AT 400 WORDS)