Tumor necrosis factor α (TNFα), an early cytokine produced by activated macrophages, plays an essential role in normal and pathological inflammatory reactions. The excessive production of TNFα is prevented by the so-called 'macrophage-deactivating factors.' This study examines the role of two structurally related neuropeptides, the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating peptide (PACAP), as inhibitors of TNFα. Both VIP and PACAP inhibit TNFα production from lipopolysaccharide-stimulated RAW 246.7 cells in a dose- and time-dependent manner. Although the activated cells express mRNA for all three VIP/PACAP receptors, agonist and antagonist studies indicate that the major receptor involved is VIP1R. VIP/PACAP inhibit TNFα gene expression by affecting both NF-kB binding and the composition of the cAMP responsive element binding complex (CREB/c-Jun). Two transduction pathways, a cAMP-dependent and a cAMP- independent pathway, are involved in the inhibition of TNFα gene expression and appear to differentially regulate the transcriptional factors involved. Because TNFα plays a central role in various inflammatory diseases such as endotoxic shock, multiple sclerosis, cerebral malaria, and various autoimmune conditions, the down-regulatory effect of VIP/PACAP may have a significant therapeutic potential.