Tumor necrosis factor-α (TNF-α) and γ-interferon (IFN-γ) cooperate during a variety of biological responses and ultimately synergistically enhance the expression of genes involved in immune and inflammatory responses. Recently, we demonstrated that IFN-γ can significantly potentiate TNF-α-induced nuclear factor (NF)-κB nuclear translocation in neuronal derived and endothelial cell lines. The mechanism by which these two cytokines exert their synergistic effect on NF-κB involves the de novo degradation of the NF-κB inhibitor, IκBβ. The double-stranded RNA- dependent kinase PKR is IFN-inducible and has been implicated in the activation of NF-κB; therefore, we examined the possibility that PKR may play a role in the synergistic activation of NF-κB during TNF-α/IFN-γ cotreatment. The PKR inhibitor 2-aminopurine (2-AP) inhibited TNF-α/IFN-γ- induced NF-κB nuclear translocation in neuronal derived cells but not in endothelial cells. The induced degradation of IκBβ, which is normally observed upon TNF-α/IFN-γ cotreatment, was blocked completely by 2-AP in neuronal derived cells. Also, 2-AP treatment or overexpression of a catalytically inactive PKR inhibited the TNF-α/IFN-γ-induced synergistic activation of κB-dependent gene expression. Our results suggest that the signal generated by IFN-γ, during TNF-α/IFN-γ cotreatment may require PKR to elicit enhanced NF-κB activity, and this signal may affect the stability of the IκBβ protein.