IL-12(p70), a heterodimer composed of two subunits (p35 and p40), is a key cytokine for Th1 mediated inflammatory responses. We dissected the role of IL-12 in the development of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis by studying mice deficient in IL-12p40, IL-12p35, or IL-12Rbeta1. TNBS-treated IL-12Rbeta1(-/-) and IL-12p35(-/-) mice developed only a mild disease associated with low level IL-18 expression in IL-12p35(-/-) mice. In contrast, IL-12p40(-/-) mice developed more severe colitis than wild-type mice associated with high level colonic IL-18 expression. Administration of IL-12p40 neutralizing mononuclear antibody dramatically increased pathology in IL-12p35(-/-) mice similar to disease scored in IL-12p40(-/-) mice. Numbers of IFN-gamma-producing cells infiltrating the lamina propria were comparably augmented in the different groups of IL-12-mutant and wild-type mice. These results demonstrate that IL-12p40, in contrast to IL-12p70, inhibits TNBS-induced colitis and IL-18 expression independent of IFN-gamma.