To explore the mechanism by which morphine promotes the incidence of HIV infection, we evaluated the regulatory role of morphine on the interferon-γ (IFN-γ) promoter in activated T cells from wild type and μ-opioid receptor knockout mice. Our results show that morphine inhibited anti-CD3/CD28-stimulated IFN-γ promoter activity in a dose-dependent manner. Chronic morphine treatment of T cells increased intracellular cAMP. To evaluate the role of cAMP in morphine's modulatory function, the effects of dibutyryl cyclic AMP and forskolin were investigated. Both dibutyryl cyclic AMP and forskolin treatment inhibited IFN-γ promoter activity. Treatment with pertussis toxin, but not with a protein kinase A inhibitor, antagonized morphine's inhibitory effects. Morphine inhibited phosphorylation of ERK1/2 and p38 MAPK; in addition, morphine treatment in the presence of either ERK1/2 or p38 MAPK inhibitor (PD98059 or SB203580) resulted in an additive inhibition of IFN-γ promoter activity. The transcription factor activator protein-1, NFκB, and nuclear factor of activated T cells (NFAT) were negatively regulated by morphine. Overexpression of NFκB p65 rescued the inhibitory effect of morphine on IFN-γ promoter activity. However, only when NFATc1 was co-overexpressed with c-fos was the inhibitory effect of morphine on IFN-γ promoter counteracted. The inhibitory effects of morphine were not observed in T cells obtained from μ-opioid receptor knockout mice, suggesting that morphine modulation of IFN-γ promoter activity is mediated through the μ-opioid receptor. In summary, our data indicate that morphine modulation of IFN-γ promoter activity is mediated through two distinct cAMP-dependent pathways, the NFκB signaling pathway and the ERK1/2, p38 MAPK, AP-1/N-FAT pathway.