The signaling pathways leading to extracellular signal-regulated kinase (ERK) activation in formyl-methionylleucyl-phenylalanine (fMLP)-stimulated rat neutrophils were examined. fMLP-stimulated ERK activation based on immunoblot analysis with antibodies against the phosphorylation form of ERK was attenuated by the pretreatment of cells with pertussis toxin but not with a dual cyclo-oxygenase/lipoxygenase inhibitor BW755C. Exposure of cells to the tyrosine kinase inhibitor genistein, phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002, or protein kinase C (PKC) inhibitors Go «6976, Go «6983, and GF109203X inhibited fMLP-stimulated ERK phosphorylation in a concentration-dependent manner. In addition, both the phospholipase C (PLC) inhibitor U73122 and the Ca 2 chelator BAPTA attenuated ERK activation. These results indicate that G iao protein, tyrosine kinase, PI3K, PKC, and PLC/Ca 2 , but not arachidonate metabolites, act upstream of fMLP-stimulated ERK activation.z 1999 Federation of European Biochemical Societies.