The responses of human neutrophils (PMN) involve reorganization and phosphorylation of cytoskeletal components. We investigated the translocation of protein kinase C (PKC) isoforms to PMN cytoskeletal (Triton-insoluble) fractions, in conjunction with activation of the respiratory burst enzyme NADPH oxidase. In resting PMN, PKC-δ (29%) and small amounts of PKC-α (0.6%), but not PKC-βII, were present in cytoskeletal fractions. Upon stimulation with the PKC agonist PMA, the levels of PKC-α, PKC-βII, and PKC-δ increased in the cytoskeletal fraction, concomitant with a decrease in the noncytoskeletal (Triton-soluble) fractions. PKC-δ maximally associated with cytoskeletal fractions at 160 nM PMA and then declined, while PKC-α and PKC-βII plateaued at 300 nM PMA. Translocation of PKC-δ was maximal by 2 min and sustained for at least 10 min. Translocation of PKC-α and PKC-βII was biphasic, plateauing at 2–3 min and then increasing up to 10 min. Under maximal stimulation conditions, PKC isoforms were entirely cytoskeletal associated. Translocation of the NADPH oxidase component p47 phox to the cytoskeletal fraction correlated with translocation of PKC-α and PKC-βII, but not with translocation of PKC-δ. Oxidase activity in cytoskeletal fractions paralleled translocation of PKC-α, PKC-βII, and p47 phox. Stimulation with 1, 2-dioctanoylglycerol resulted in little translocation of PKC isoforms or p47 phox, and in minimal oxidase activity. We conclude that conventional PKC isoforms (PKC-α and/or PKC-βII) may regulate PMA-stimulated cytoskeletal association and activation of NADPH oxidase. PKC-δ may modulate other PMN responses that involve cytoskeletal components.