IL-12-induced IFN-γ production is essential for clearance of Yersinia enterocolitica infection. Similar to IL-12, the recently described cytokine IL-18 (IFN-γ-inducing factor) is produced by macrophages and induces IFN-γ production in spleen cells. Therefore, we have investigated the role of IL-18 in Yersinia infection of mice. Heat-killed yersinia-triggered IL-18-promoted IFN-γ production of splenocytes was predominantly dependent on endogenous IL-12 production, whereas IL-12-promoted IFN-γ production was not IL-18 dependent. IL-18-induced IFN-γ production was to a higher degree dependent on IFN-γR-mediated mechanisms and in synergism with IL-2 resulted in at least fivefold higher IFN-γ levels as compared with the combination of IL-12 plus IL-2. Analysis of the effect of IL-18 on IL-12 production of LPS-stimulated peritoneal macrophages revealed that IL-18 decreased LPS-induced IL-12 production, indicating that IL-18 might be involved in negative regulation of IL-12 production. In vivo studies revealed that Yersinia-resistant C57BL/6 mice expressed fourfold higher IL-18 mRNA levels than did susceptible BALB/c mice. Administration of anti-IL-18 Abs caused a 100-to 1000-fold increase in bacterial counts in the spleen of infected mice but did not change IFN-γ production levels. Taken together, our data demonstrate that IL-18 is involved in regulation of cytokine production during the early phase of bacterial infections as well as in clearance of Yersinia infection.