The possibility that endogenous monohydroxy-eicosatetraenoic acids (HETEs) derived from the lipoxygenation of arachidonic acid might serve a role in human neutrophil migration was examined by studying the effects of depletion of the intracellular HETEs on random migration and chemotaxis. The intracellular contents of approximately 2000 ng of 11-HETE and 500 ng of 5-HETE per 10(8) neutrophils are distributed preferentially in the cellular membranes and are increased by specific chemotactic factors. The depletion of intracellular HETEs that resulted from pre-incubating, washing and resuspending neutrophils in 3-20 microM 5,8,11,14-eicosatetraynoic acid (TYA), an inhibitor of lipoxygenase and cyclooxygenase activity, or in 5-10 microM nordihydroguaiaretic acid (NDGA), a selective inhibitor of lipoxygenase activity, was associated with suppression of neutrophil random migration and chemotaxis to several stimuli without evidence of cytotoxicity. Maximal suppression of migration was achieved by a 30-60 min preincubation with the inhibitors, a time-course analogous to that required for optimal depletion of the endogenous HETEs. In contrast, inhibitors of cyclooxygenase activity enhanced random migration and, to a lesser extent, chemotaxis. The inhibition of migration achieved by pre-incubating and maintaining the neutrophils in TYA or NDGA was fully reversed either by washing and resuspending the neutrophils in buffer or by the addition of purified neutrophil 5-HETE in quantities as small as 20 ng/2 x 10(6) neutrophils for random migration and 0.8 ng/2 x 10(6) neutrophils for chemotaxis, while the addition of 11-HETE was less effective. The relationship of the intracellular concentrations of endogenous HETEs to neutrophil migration is consistent with a potential role of the HETEs as cellular mediators.