Vasoactive intestinal peptide (VIP), a neuropeptide present in the lymphoid microenvironment, and the structurally related pituitary adenylate cyclase-activating polypeptide (PACAP) act as potent anti-inflammatory agents that inhibit the function of activated macrophages and TH cells. Previous reports showed that VIP/PACAP inhibit IL-6 and TNF-alpha production in LPS-stimulated macrophages. The present study reports on the effect of VIP/PACAP on IL-10 production. Although VIP/PACAP do not induce IL-10 by themselves, they enhance IL-10 production in LPS-stimulated macrophages. The specific VPAC1 receptor mediates the stimulatory effect of VIP/PACAP, and cAMP is the major second messenger involved. VIP/PACAP increase IL-10 mRNA in LPS-stimulated cells, and the effect of transcriptional and protein synthesis inhibitors indicates de novo IL-10 production. Electromobility shift assays show that VIP/PACAP induce an increase in nuclear cAMP response element (CRE)-binding complexes, with CRE binding protein as the major active component. Treatments with either a VPAC1 antagonist or a protein kinase A inhibitor abolish IL-10 stimulation and, concomitantly, the increase in CRE binding. Effects similar to the in vitro stimulation of IL-10 were obtained in vivo in mice treated with LPS and VIP or PACAP. The neuropeptides induce increased levels of IL-10 in both serum and peritoneal fluid, and increased expression of the IL-10 mRNA in peritoneal exudate cells. The stimulation of IL-10 production in activated macrophages represents a novel anti-inflammatory activity of VIP and PACAP, which presumably acts in vivo in conjunction with the inhibition of proinflammatory cytokines such as IL-6 and TNF-alpha to reduce the magnitude of the immune response.