Vasoactive intestinal peptide (VIP), a neuropeptide present in the peptidergic innervation of lymphoid organs and expressed in thymocytes and peripheral lymphocytes, modulates cytokine expression in T lymphocytes. VIP down-regulates the expression of IL-2 and IL-10 mRNA in T cells stimulated through the TCR-associated CD3 complex. In contrast, IL-4 production is inhibited at a post-transcriptional level. In this study, we investigate the molecular mechanisms involved in the inhibition of IL-4 production by VIP. At the protein level, the time courses for IL-2 and IL-4 inhibition by VIP are different, with IL-4 being affected approximately 24 h later than IL-2. Northern blots and competitive reverse-transcription PCR analysis confirm the post-transcriptional inhibition of IL-4 production in both murine spleen cells and thymocytes activated through the CD3 complex. VIP does not affect IL-4 secretion, and does not induce a rapid IL-4 reuptake. Exogenous rIL-2 completely reverses the inhibitory effect of VIP, suggesting that VIP inhibits IL-4 production indirectly as a consequence of IL-2 inhibition. Studies regarding the newly synthesized IL-4 protein show that although VIP and exogenous IL-2 do not affect the rate of IL-4 synthesis, VIP reduces significantly the stability of the newly synthesized IL-4 protein, and exogenous IL-2 can restore it to the levels observed in activated cells in the absence of VIP. These results explore the molecular mechanisms involved in the neuroendocrine regulation of cytokine production, and support the idea that neuropeptides released or produced in the local lymphoid microenvironment may participate in the intricate cytokine network controlling local immune responses.