We have described conditions by which MHC class II (I-A) glycoproteins can be induced to be differentially expressed after treatment of macrophages with rIFN-gamma. Treatment of macrophages from BCG-resistant mice with 1 U of rIFN-gamma induced transient I-A expression that decayed in the presence of cycloheximide. Subsequent treatment of these macrophages with 100 U of rIFN-gamma induced the persistence of I-A that was not affected by cycloheximide. The aim of this investigation was to define, by pharmacologic intervention, the second signals that resulted in the induction of persistence of I-A. Treatment of the macrophages that transiently expressed I-A with PMA resulted in the induction of persistence. When we compared the effect of different protein kinase C (PKC) inhibitors with the induction of persistence by rIFN-gamma, we found that H-7 blocked the induction of persistence only when added before or at the same time as the addition of a high dose of rIFN-gamma. In contrast, the addition of staurosporine to macrophages as late as 2 h after treatment with high doses of rIFN-gamma inhibited the induction of I-A persistence. The addition of a high dose of rIFN-gamma to macrophages previously treated with a low dose of rIFN-gamma resulted in the synergistic activation of PKC. The effect of H-7 and of staurosporine on the activation of PKC activity coincided with the effect of these inhibitors on the induction of persistent I-A expression. Tyrosine kinase inhibitors genistein and herbimycin did not affect the induction of I-A persistence nor of PKC activation. Antibody to the IFN-gamma receptor inhibited PKC activation. Finally, the addition of the high dose of rIFN-gamma to macrophages from BALB/c.Bcgs mice, previously treated with the low dose of rIFN-gamma, failed to activate high levels of PKC activity attained after similar treatment of macrophages from BALB/c.Bcgr mice. One effect of the Bcg gene may be to regulate the activation of PKC activity.