We previously reported that the stimulation of human blood monocytes with IFN-gamma induces the binding of PU.1 to the gp91(phox) promoter and the consequent expression of gp91(phox). In this study, we show that the effect of IFN-gamma is reproduced by the serine phosphatase inhibitor, okadaic acid, and this suggests that serine kinases could be involved in gp91(phox) expression. We also show that IFN-gamma induces the serine/threonine phosphorylation of PU.1 in cultured monocytes. This phosphorylation, as well as the IFN-gamma-induced PU.1 binding and gp91(phox) protein synthesis, is slightly affected by the casein kinase II inhibitor, daidzein, but is abrogated by the protein kinase C (PKC) -alpha and -beta inhibitor, Go6976, and by synthetic peptides with sequences based on the endogenous pseudosubstrate region of the classical PKC alpha and beta isoforms. In contrast, peptides reproducing the pseudosubstrate region of PKC epsilon were without effect. Moreover, we found that the treatment of monocytes with IFN-gamma induces the nuclear translocation and the activation of PKC alpha and beta I, but not of PKC beta II, and that the IFN-gamma-induced phosphorylation of PU.1 was greatly reduced by LY333531, a selective inhibitor of PKC beta isoforms. Finally, nuclear run-on assays demonstrated that while the PKC inhibitors, Go6976 and LY333531, decrease the IFN-gamma-induced gp91(phox) transcription, the serine phosphatase inhibitor, okadaic acid, enhances the gp91(phox) gene transcription. Our results indicate that in cultured monocytes, IFN-gamma induces the binding of PU.1 to the gp91(phox) promoter and the expression of gp91(phox) by phosphorylation of PU.1 via activation of PKC alpha and/or beta I.