We previously reported that the stimulation of human blood monocytes with IFN-γ 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-γ 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-γ induces the serine/threonine phosphorylation of PU. 1 in cultured monocytes. This phosphorylation, as well as the IFN-γ-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)-α and-β inhibitor, Go6976, and by synthetic peptides with sequences based on the endogenous pseudosubstrate region of the classical PKC α and β isoforms. In contrast, peptides reproducing the pseudosubstrate region of PKC ε were without effect. Moreover, we found that the treatment of monocytes with IFN-γ induces the nuclear translocation and the activation of PKC α and βI, but not of PKC βII, and that the IFN-γ-induced phosphorylation of PU. 1 was greatly reduced by LY333531, a selective inhibitor of PKC β isoforms. Finally, nuclear run-on assays demonstrated that while the PKC inhibitors, Go6976 and LY333531, decrease the IFN-γ-induced gp91 phox transcription, the serine phosphatase inhibitor, okadaic acid, enhances the gp91 phox gene transcription. Our results indicate that in cultured monocytes, IFN-γ 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 α and/or βI.