Injecting semiallogeneic CAF1 spleen into BALB/c newborn mice renders mice tolerant, and the majority of mice show prolonged survival of tolerogen-bearing A/J skin grafts. Moreover, graft survival is associated with enhanced Th2 cytokine responses and graft rejection with Th1 cytokine responses. To further delineate the mechanisms of tolerance, we evaluated CTL responses and found that 74% of neonatal primed mice failed to generate A/J-specific CTL responses, as determined by standard CTL assays and pTc3 frequency analyses. CTL unresponsiveness coexisted with an enhanced tolerogen-specific Th2 memory cytokine profile; spleen cells from neonatal primed mice secreted more interleukin (IL)-4 and less IL-2 and interferon (IFN)-gamma in MLR cultures compared with either adult primed or naive controls. We therefore examined the hypothesis that enhanced Th2 cytokine levels prevent the generation of tolerogen-specific CTL. Adding neutralizing antibodies to IL-4 and IL-10 recovered IFN-gamma production in vitro but not A/J-specific CTL response. In addition, CD4 cells from neonatal primed mice provided help for primary or secondary CD8 CTL generation, which suggests that the enhanced Th2 cytokine profile does not actively suppress CTL generation. Furthermore, CD4 cells from adult primed mice failed to restore the A/J-specific CD8 CTL generation of neonatal primed mice. The results show that failure to develop A/J-specific CTL reaction occurs without suppression by the enhanced Th2-type responses and imply that either deletion or anergy mechanisms block CTL generation. Therefore, neonatal alloantigen exposure not only shifts the development of alloreactive CD4 cells toward Th2, but also blocks development of alloreactive CD8 CTL in this strain combination.