Roles of protein kinase C and protein tyrosine kinase in the activation of neutrophil respiratory burst, degranulation and elevation of cytosolic Ca²⁺ in platelet-activating factor (PAF)-stimulated neutrophils were investigated. Superoxide and H₂O₂ production and myeloperoxidase and acid phosphatase release in PAF-stimulated neutrophils were inhibited by protein kinase C inhibitors, staurosporine and H-7 and protein tyrosine kinase inhibitors, genistein and tyrphostin. The PAF-induced elevation of [Ca²⁺]_i2+ release and Mn²⁺ influx in PAF-stimulated neutrophils. Genistein and methyl-2,5-dihydroxycinnamate inhibited Mn²⁺ influx induced by PAF, whereas their effects on intracellular Ca²⁺ release were not detected. In neutrophils preactivated by PMA, the stimulatory effect of PAF on the elevation of [Ca²⁺]_i was reduced. Protein kinase C and protein tyrosine kinase may be involved in respiratory burst, lysosomal enzyme release and Ca²⁺ mobilization in PAF-stimulated neutrophils. The elevation of [Ca²⁺]_i2+ release and Ca²⁺ influx which are differently regulated by protein kinases. Preactivation of protein kinase C appears to attenuate the stimulatory action of PAF on intracellular Ca²⁺ mobilization.