Adenosine is an important inhibitory neuromodulator in the cerebral cortex, yet it remains unclear how extracellular adenosine concentrations are regulated. Recently, it has been shown that beta-adrenergic receptor stimulation in rat cortical cultures causes the accumulation of extracellular adenosine derived by enzymatic hydrolysis from adenosine cyclic monophosphate (cAMP) transported into the extracellular space. In this study we show that vasoactive intestinal peptide (VIP), in addition to activating adenylyl cyclase and promoting the accumulation of intracellular cAMP in rat cortical cultures, also causes transport of cAMP and accumulation of extracellular adenosine. We further show that the extracellular accumulation of adenosine in response to VIP can be blocked by inhibition of cAMP transport, cyclic nucleotide phosphodiesterase activity, and 5'-nucleotidase, indicating that extracellular cAMP is the source of the adenosine. Cyclic AMP transport may be a general mechanism by which a variety of neuromodulators that act upon receptors coupled to adenylyl cyclase might regulate extracellular adenosine levels and thereby inhibitory tone in the cerebral cortex.