Vasoactive intestinal peptide (VIP), a neuropeptide present in the peptidergic innervation of lymphoid organs and expressed in thymocytes and peripheral lymphocytes has been previously reported to modulate cytokine expression in T lymphocytes. In this study, we investigated the effects of VIP and of the structurally related neuropeptide PACAP-38 on the expression of IL-10 in murine lymphocyte cultures. Both neuropeptides inhibit IL-10 production by spleen cells or thymocytes activated via the TCR-associated CD3 complex in a similar dose-response manner. The inhibition is specific, presumably mediated through the VIP-R1, and maximum inhibitory levels are achieved within the first 5 to 15 min of exposure to VIP or PACAP-38. CD4+ T cells function as direct cellular targets for the two neuropeptides. The fact that VIP, PACAP-38, and forskolin, all known cAMP inducers, also inhibit IL-10 production, suggests the participation of cAMP in signal transduction. VIP and PACAP-38 regulate transcriptional expression of IL-10, since IL-10 steady state mRNA levels are significantly reduced by treatment with the two neuropeptides. These results expand the range of neuroendocrine-regulated cytokines and support the idea that neuropeptides such as VIP and PACAP, which are released or produced in the local lymphoid microenvironment and specifically modulate the expression of various cytokines, may participate in the intricate cytokine network controlling local immune responses.