The purpose of the present work was to investigate the mechanism underlying the inhibitory action of rebamipide on superoxide anion (O2) production induced by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) in human neutrophils. Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), a product of phosphoinositide 3-OH-kinase (PI 3-kinase) accumulated in response to fMLP and this accumulation was well correlated with O2 production in human neutrophils. Rebamipide inhibited PIP(3) production in parallel with the inhibition of fMLP-induced O2 production. PI 3-kinase activity in anti-PI 3-kinase p85 immunoprecipitates was not affected by the presence of rebamipide, therefore rebamipide did not have a direct inhibitory action on PI 3-kinase activity. Since rebamipide had no inhibitory effect on O2 production induced by NaF, a direct activator of G protein, the target of the inhibitory action of rebamipide appears to be a component of the signal transduction pathway upstream of G protein. Scatchard analysis of [3H]fMLP binding to human neutrophil membrane revealed that rebamipide increased the K(D) value of [3H]fMLP without altering the number of [3H]fMLP binding sites, suggesting that rebamipide has a competitive antagonistic action against the fMLP-receptor. The competitive antagonistic action was further confirmed by the finding that rebamipide caused a parallel shift to the right in the dose-response curve of O2 production induced by fMLP. These results provide evidence that the competitive inhibitory action of rebamipide on the fMLP-receptor plays a main role in its inhibitory action on fMLP-induced O2 production.