The murine macrophage cell line, J774,when activated with interferon-gamma (IFN-gamma), expressed high level of inducible nitric oxide synthase (iNOS) and bound significantly more [H-3]-phorbol-dibutyrate (PBu(2)) compared to non-activated cells. The increased PBu(2) binding to the particulate fraction of the cells is a measure of activation and translocation of protein kinase C (PKC). Both the expression of iNOS and the enhanced PBu(2) binding in the activated J774 cells were significantly inhibited by the pretreatment of the cells with murine recombinant interleukin-4 (IL-4). Stimulation of J774 cells by IFN-gamma and lipopolysaccharide results in the translocation predominantly of the epsilon isoform of PKC (PKC-epsilon), and this is inhibited by IL-4. The inhibition of PKC activation was also evident by measuring the PKC activity in the cytosolic fraction of the IL-4-treated cells. Activated 5774 cells pretreated with IL-4 or a PKC-specific inhibitor (RO31-8220) failed to express mRNA of iNOS analyzed by PCR. These results, therefore, suggest that the inhibition of nitric oxide synthesis in activated murine macrophages by IL-4 is at the transcriptional level and may involve the inhibition of the activation of PKC-epsilon.