To define a possible role of the enzyme phosphatidylinositol 3-kinase (PI 3-kinase) in motile functions of neutrophils, we have used a potent inhibitor of this enzyme, [1S-(1alpha,6b alpha,9a beta,11alpha,11bbeta)]-1-(acetyloxy)-1,6b,7,8,9a,10,11 ,11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3,2-de]indeno [4,5-h]-2-benzopyran-3,6,9-trione (wortmannin). Wortmannin markedly attenuated chemotactic peptide-induced development of polarity, locomotion and increases in cytoskeletal actin and alpha-actinin in human neutrophils at low, nM, concentrations (ED50 = 4-40 nM; 0.4-3 pmol/10(6) cells). The increase in cytoskeletal actin induced by phorbol-12-myristate-13-acetate in contrast was not affected by wortmannin (18 pmol/10[6] cells). Moreover, the increase in total F-actin induced by an incubation for 1 min with chemotactic peptide was much less sensitive to wortmannin than increases in cytoskeletal actin; 80 pmol/10(6) cells were necessary for half-maximal inhibition. Wortmannin thus appears to primarily affect F-actin organization, rather than polymerization. Inhibition of development of polarity by wortmannin correlated with inhibition of production of phosphatidylinositol 3,4,5-trisphosphate. According to our findings, activation of a wortmannin-sensitive target, very likely PI 3-kinase, is required for optimal chemotactic peptide-induced neutrophil motility.