Leukotrienes (LTs) are lipid mediators that participate in inflammatory diseases and innate immune function. We sought to investigate the importance of LTs in regulating the microbicidal activity of alveolar macrophages (AMs) and the molecular mechanisms by which this occurs. The role of LTs in enhancing AM microbicidal activity was evaluated pharmacologically and genetically using in vitro challenge with Klebsiella pneumoniae. Exogenous LTs increased AM microbicidal activity in a dose- and receptor-dependent manner, and endogenous production of LTs was necessary for optimal killing. Leukotriene B4 (LTB4) was more potent than cysteinyl LTs. An important role for nicotinamide adenine dinucleotide (NADPH) oxidase in LT-induced microbicidal activity was indicated by the fact that bacterial killing was abrogated by the NADPH oxidase inhibitor diphenyleneiodonium (DPI; 10 microM) and in AMs derived from gp91phox-deficient mice. By contrast, LT-induced microbicidal activity was independent of the generation of nitric oxide. LTs increased H2O2 production, and LTB4 was again the more potent agonist. Both classes of LTs elicited translocation of p47phox to the cell membrane, and LTB4 induced phosphorylation of p47phox in a manner dependent on protein kinase C-delta (PKC-delta) activity. In addition, the enhancement of microbicidal activity by LTs was also dependent on PKC-delta activity. Our results demonstrate that LTs, especially LTB4, enhanceAM microbicidal activity through the PKC-delta-dependent activation of NADPH oxidase.