Leukotrienes (LTs) are known to be produced by macrophages when challenged with Leishmania, but it is not known whether these lipid mediators play a role in host defense against this important protozoan parasite. In this study, we investigated the involvement of LTs in the in vitro and in vivo response to Leishmania amazonensis infection in susceptible (BALB/c) and resistant (C3H/HePAS) mice. Pharmacologic or genetic deficiency of LTs resulted in impaired leishmanicidal activity of peritoneal macrophages in vitro. In contrast, addition of LTB4 increased leishmanicidal activity and this effect was dependent on the BLT1 receptor. LTB4 augmented NO production in response to L. amazonensis challenge, and studies with a NO synthesis inhibitor revealed that NO was critical for the enhancement of macrophage leishmanicidal activity. Interestingly, macrophages from resistant mice produced higher levels of LTB4 upon L. amazonensis challenge than did those from susceptible mice. In vivo infection severity, as assessed by footpad swelling following s.c. promastigote inoculation, was increased when endogenous LT synthesis was abrogated either pharmacologically or genetically. Taken together, these results for the first time reveal an important role for LTB4 in the protective response to L. amazonensis, identify relevant leishmanicidal mechanisms, and suggest that genetic variation in LTB4 synthesis might influence resistance and susceptibility patterns to infection.