Healthy human peripheral neutrophils (HPPMN) are not primed, and have weak responses to stimuli which activate HPPMN through their membrane receptors. Recombinant human tumor necrosis factor-α (rHuTNF) and recombinant granulocyte colony stimulation factor (rG-CSF) primed HPPMN. Superoxide (O^&minusd;_2) generation by formylmethionyl-leucylphenylalanine (FMLP) or opsonized zymosan (OZ) was enhanced by these primers. However, O^&minusd;_2 generation induced by phorbol myristate acetate (PMA) or dioctanoylglycerol (DOG) was not enhanced by the primers. Receptor mediated O^&minusd;_2 generation in rHuTNF primed neutrophils was inhibited by the genistein or alpha-cyan0-3-ethoxy-4-hydroxy-5phenylthiomethylcinnamamide (ST 638), inhibitors of tyrosine kinase (TK). But it was enhanced by 1-(5-isoquinoline sulfonyl)-3-methyl-piperazine (H-7) or staurosporine, inhibitors of Ca^<++>_ and phospholipid-dependent protein kinase (PKO, in their concentration dependent manner. On the contrary, O^&minusd;_2 generation of HPPMN at higher concentration of PMA or DOG was rather stimulated by genistein or ST 638 and was inhibited by H-7 or staurosporine. These results suggest that protein kinases participates in the NADPH-oxidase activation of neutrophils and that two pathways exist in the NADPH-oxidase activation : one in PMA- or DOG-stimulated PKC-dependent pathway and the other in FMLP-stimulated TK-dependent pathway. Moreover, it suggests that TK might be involved in the reaction of neutrophil priming with various ligands.