The present report provides evidence for a novel function for the neuropeptide vasoactive intestinal peptide (VIP). We demonstrate that VIP increases the cholinergic and the noradrenergic properties of cultured chick sympathetic neurons without changing neuronal survival and metabolism. VIP induces a 10- to 15-fold increase in the activity of choline acetyltransferase and an approximately twofold increase in the activity of tyrosine hydroxylase. Forskolin, an activator of adenylate cyclase, mimics all the effects of VIP on these cells. In addition, the effects of forskolin and VIP at optimal concentrations are not additive. Furthermore, VIP induces a rapid increase in the intracellular cAMP levels. Thus VIP acts via a cAMP-dependent pathway to enhance the cholinergic and noradrenergic properties of cultured chick sympathetic neurons.