Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) induces respiratory burst (O-2 generation) in permeabilized human neutrophils. The signal pathway from GTPgammaS to the enzyme responsible for O-2 generation (NADPH oxidase) is not well defined. To elucidate the signaling pathway activated by GTPgammaS, we used selective inhibitors to test for the involvement of several enzymes, comparing the effects of these inhibitors on fMet-Leu-Phe (fMLP) activation. GTPgammaS-induced respiratory burst was not influenced by genistein, a selective inhibitor of tyrosine kinase, while fMLP-induced response was completely abolished. The respiratory burst by GTPgammaS was efficiently inhibited by the protein kinase C inhibitor GF109203X even more than fMLP activation. The mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 showed a partial inhibition of both GTPgammaS and fMLP activation. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, completely blocked fMLP activation, but had no effect on the GTPgammaS-induced respiratory burst. Using U73122, phospholipase C is shown to be essential in GTPgammaS signaling as well as fMLP signaling. Butanol blocked fMLP signaling but not GTPgammaS signaling, indicating that only fMLP activation involves phospholipase D. These results suggest that there are several differences between GTPgammaS- and fMLP-induced activation, but both activators share a common pathway including phospholipase C, protein kinase C, and MAPK kinase.