Interleukin-18 (IL-18) is a proinflammatory cytokine that plays an important role in natural killer cell activation and the T helper 1 (Th1) cell response, particularly in collaboration with IL-12. Since Th1 cells play a pivotal role in the host defense against infection with intracellular microbes, such as Leishmania major, we investigated whether IL-18 is critically involved in protection against L. major infection by activation of Th1 cells. We administered IL-12 and/or IL-18 daily to L. major-susceptible BALB/c mice. Neither IL-12 (10 ng/mouse) nor IL-18 (1,000 ng/mouse) induced wound healing, while daily injection of IL-12 and IL-18 during the first week after infection strongly protected the mice from footpad swelling by induction and activation of Th1 cells. Furthermore, these mice acquired protective immunity. We also investigated a protective role of endogenous IL-18 by using anti-IL-18 antibody-treated C3H/HeN mice (an L. major-resistant strain) or IL-18 deficient (IL-18(-/-)) mice with a resistant background (C57BL/6). We found that in the absence of endogenous IL-18, these mice showed prolonged footpad swelling as well as diminished nitric oxide production. However, daily injection of IL-18 into IL-18(-/-) mice corrected their deficiencies, suggesting that these mice have Th1 cells that produce gamma interferon (IFN-gamma) in response to IL-18. Indeed, these mice had normal levels of Th1 cells. Thus, IL-18 is not responsible for inducing Th1 cells but participates in host resistance by its action in stimulating Th1 cells to produce IFN-gamma. Our results also indicate the high potentiality of IL-18 as a useful reagent for treatment as well as prevention against reinfection.