Human neutrophils maximally stimulated with the optimal concentration (100 ng/ml) of phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), for 5 min at 37°C did not respond with superoxide (O − 2) release to the later addition of PMA itself or the Ca 2+ ionophore ionomycin. However, these cells did respond with enhanced release of O − 2 to the later addition of N- formyl-methionyl-leucyl-phenylalanine (FMLP) or concanavalin A (Con A). In these PMA-pretreated cells, an increase in cytoplasmic free Ca 2+ ([Ca 2+] i) induced by ionomycin was unaffected, whereas that induced by FMLP was inhibited by 50–60% and that induced by Con A was completely abolished. A 42-kDa protein was predominantly and consistently tyrosine-phosphorylated by FMLP, PMA and ionomycin with the different kinetics according to the stimuli. The dose-response curves showed that tyrosine phosphorylation and O − 2 release were stimulated in parallel by PMA, whereas tyrosine phosphorylation and an increase in [Ca 2+] i, but not O − 2 release, were stimulated in parallel by FMLP or ionomycin. The potency of inducing tyrosine phosphorylation was ionomycin > FMLP = PMA , whereas the potency of triggering of O − 2 release was PMA > ionomycin = FMLP . UCN-01, a PKC inhibitor, inhibited O − 2 release and tyrosine phosphorylation induced by PMA, but not by FMLP or ionomycin. In contrast, pertussis toxin inhibited O − 2 release and tyrosine phosphorylation induced by FMLP, but not by PMA. Tyrosine kinase inhibitors (erbstatin and genistein) inhibited O − 2 release induced by FMLP, but not by PMA. However, both tyrosine kinase inhibitors did not impair FMLP- or PMA-induced tyrosine phosphorylation of a 42-kDa protein. Increased tyrosine phosphorylation of a 42-kDa protein was also detected in immature myeloid cells (HL-60 cells) stimulated by PMA, but not by ionomycin. These findings suggest that FMLP and Con A trigger the respiratory burst in human neutrophils by activating the definite pathway which include other signals than activation of PKC and an increase in [Ca 2+] i; tyrosine phosphorylation of a 42-kDa protein is induced by the PKC-dependent and independent mechanisms according to the stimuli, and the PKC-independent and ionomycin-sensitive mechanism is inoperative in HL-60 cells; and tyrosine phosphorylation of a 42-kDa protein is unlikely to be causally related to activation of the respiratory burst.