IL-12p70, a 70-to 75-kDa heterodimer consisting of disulfide-bonded 35-kDa (p35) and 40-kDa (p40) subunits, enhances Th1 development primarily by its ability to induce IFN-γ production by NK and Th1 cells. Although homodimers of the p40 subunit of IL-12 are potent IL-12 receptor antagonists in some systems, we have reported that p40 homodimer may accentuate alloreactive CD8+ Th1 function. To test the role of endogenously produced p40 in alloimmunity, Th1 development was assessed in either IL-12 p35 knockout (p35−/−) mice, the cells of which are capable of secreting p40, or p40 knockout (p40−/−) mice. Compared with IL-12 wild-type controls, splenocytes obtained from both p35−/− and p40−/− mice produced markedly less IFN-γ after in vitro stimulation with Con A or alloantigens. Interestingly, in vivo-sensitized Th1 were detected in both p35−/− and p40−/− cardiac allograft recipients. However, in vivo Th1 development was enhanced in p35−/− recipients compared with p40−/− animals, suggesting that endogenous p40 produced in p35−/− mice may stimulate alloreactive Th1. Indeed, neutralizing endogenous p40 with anti-IL-12 p40 mAb reduced Th1 development in p35−/− allograft recipients to that seen in p40−/− mice. To determine whether Th1 development that occurred in the absence of IL-12p70 and p40 required IFN-γ, p40−/− allograft recipients were treated with anti-IFN-γ mAb. Neutralizing IFN-γ did not inhibit in vivo Th1 development in p40−/− recipients and resulted in a unique pathology of rejection characterized by vascular thromboses. Collectively, these data suggest that 1) endogenous p40 may substitute for IL-12p70 in alloantigen-specific Th1 sensitization in vivo and 2) in vivo alloreactive Th1 development may occur independent of IL-12 and IFN-γ, suggesting an alternate Th1-sensitizing pathway.