Phosphorylation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase components p67phox and p47phox accompanies the assembly and activation of this enzyme complex. We have previously reported that activation of human monocytes with opsonized zymosan (ZOP), a potent stimulator of NADPH oxidase activity, results in the phosphorylation of p67phox and p47phox. In this study, we investigated the regulation of p67phox phosphorylation. Although protein kinase C (PKC)α has previously been shown to regulate NADPH oxidase activity, we found that inhibition of PKCα had no effect on p67phox phosphorylation. Our studies demonstrate that pretreatment of monocytes with antisense oligodeoxyribonucleotides specific for PKCδ or rottlerin, a selective inhibitor for PKCδ, inhibited the phosphorylation of p67phox in monocytes, and Go6976, a specific inhibitor for conventional PKCs, PKCα and PKCβ, had no such inhibitory effect. Additional studies indicate that ZOP stimulation of monocytes induces PKCδ and p67phox to form a complex. We also demonstrate that lysates from activated monocytes as well as PKCδ immunoprecipitates from activated monocytes can phosphorylate p67phox in vitro and that pretreatment of monocytes with rottlerin blocked the phosphorylation in each case. We further show that recombinant PKCδ can phosphorylate p67phox in vitro. Finally, we show that PKCδ-deficient monocytes produce significantly less superoxide anion in response to ZOP stimulation, thus emphasizing the functional significance of the PKCδ regulation of p67phox phosphorylation. Taken together, this is the first report to describe the requirement of PKCδ in regulating the phosphorylation of p67phox and the related NADPH oxidase activity in primary human monocytes.