Phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) is rapidly produced upon exposure of neutrophils to the chemoattractant N-formylmethionyl-leucylphenylalanine (fMLP), and has been proposed to act as a second messenger mediating actin polymerization and respiratory-burst activity. Here we present evidence that wortmannin, a known inhibitor of respiratory-burst activity, acts on PtdIns 3-kinase, the enzyme producing PtdInsP3 from PtdIns(4,5)P2. Pretreatment of 32P-labelled human neutrophils with 100 nM wortmannin totally abolished fMLP-mediated PtdInsP3 production, raised PtdInsP2 levels, and did not affect cellular PtdInsP and PtdIns contents. The inhibitory effect on PtdInsP3 formation in intact cells was dose-dependent, with an IC50 of approximately 5 nM. Similar results were obtained with PtdIns 3-kinase immunoprecipitated by antibodies against the p85 regulatory subunit: wortmannin totally inhibited PtdIns3P production in immunoprecipitates at concentrations of 10-100 nM (IC50 approximately 1 nM). These results illustrate the direct and specific inhibition of PtdIns 3-kinase by wortmannin. Since agonist-mediated respiratory-burst activation is most sensitive to wortmannin (IC50 = 12 nM), this suggests that agonist-mediated PtdInsP3 formation is indispensable for this cell response. Neutrophils pretreated with wortmannin develop oscillatory changes in F-actin content, but actin polymerization in response to fMLP is not inhibited. This, and the absence of PtdInsP3 under these conditions, are in agreement with a modulatory role for PtdInsP3 in cytoskeletal rearrangements, but imply that PtdInsP3 production is not a primary event triggering elongation of actin filaments in neutrophils.