We have shown that platelet-activating factor (PAF), a weak primary stimulus for neutrophil superoxide generation, synergistically enhances neutrophil oxidative responses to the tumor promoter phorbol myristate acetate (PMA). Since PMA is known to cause cytosol-to-membrane shift of calcium-activated, phospholipiddependent protein kinase (protein kinase c, PKC) in human neutrophils, we investigated the role of PAF in modifying PMA-induced PKC activation/translocation. Protein kinase activity was measured as the incorporation of "P from Y -~~P -A T P into histone H I induced by enzyme in cytosolic and particulate fractions from sonicated human neutrophils. PAF did not alter the sharp decrease in cytosolic PKC activity induced by PMA. However, in the presence of PAF and PMA, total particulate protein kinase activity increased markedly over that detected in the presence of PMA alone (144 * 9 pmoles "Pi1 07PMN/minute in cells treated with 20 ngiml PMA compared to 267 t 24 pmoles 32P in cells exposed to PMA and 1 O-6M PAF). The increase in total particulate protein kinase activity was synergistic for the two stimuli, required the presence of cytochalasin B during stimulation, and occurred at PAF concentrations of 10-7M and above. Both PKC and calcium-, phospholipid-independent protein kinase activities in whole particulate fractions were augmented by PAF as were both activities in detergent-extractable particulate subfractions. PAF did not directly activate PKC obtained from control or PMA-treated neutrophils. However, the PKC-enhancing effect of PAF was inhibited in the absence of calcium during cellular stimulation. PAF also increased particulate protein kinase activity in cells simultaneously exposed to FMLP but the effect was additive for these stimuli. These results suggest that PAF enhances PMA-induced particulate PKC activity by a calciumdependent mechanism. The enhancing effect of PAF may be directly involved in the mechanism whereby the phospholipid "primes" neutrophils for augmented oxidative responses to PMA.