Polyunsaturated fatty acids (PUFAs) and leukotriene B(4) (LTB(4)) are involved in many inflammatory and physiological conditions. The role of arachidonic acid (AA) and linoleic acid (LA) in promoting the assembly of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits is well known, but the involvement of LTB(4) and other 5-lipoxygenase (5-LO) pathway metabolites of AA in hydrogen peroxide (H(2)O(2)) production by PUFA-stimulated polymorphonuclear leukocytes (PMNs) has not been investigated. We examined this question by determining H(2)O(2) production as well as phosphorylation and membrane translocation of the p47phox subunit of NADPH oxidase. Elicited peritoneal PMNs from rats and from 5-LO-deficient or wild-type mice were pretreated with or without inhibitors of LT biosynthesis and antagonists of the receptors for LTB(4) and cysteinyl LTs for 20 min before stimulation with AA (at 5 and 20 microM) or LA (at 20 microM). PUFAs elicited H(2)O(2) production in a dose-dependent manner, and pharmacologic or genetic inhibition of LT synthesis decreased H(2)O(2) production by approximately 40% when compared with untreated controls. LTB(4) was the moiety responsible for H(2)O(2) production, as revealed by studies using receptor antagonists and its exogenous addition. LTB(4) itself also promoted p47phox phosphorylation and translocation. These results identify a heretofore unrecognized role for activation of 5-LO and subsequent production of LTB(4) in stimulation of PMN NADPH oxidase activation by PUFAs.