Adenosine is an important inhibitory neuromodulator in the CNS, yet the sources of extracellular adenosine have yet to be well characterized. In this study we show that beta-adrenergic stimulation of cortical cultures results in the extracellular accumulation of cAMP as well as adenosine, and that the extracellular adenosine derives from extracellular cAMP. The concentration dependence of isoproterenol in evoking cAMP secretion was determined by radioimmunoassay, and the EC50 for this effect was found to be approximately 100 nM. In order to investigate the effect of beta-adrenergic stimulation on the regulation of extracellular adenosine, the effect of isoproterenol in stimulating the extracellular accumulation of adenine-containing compounds was examined by HPLC. Isoproterenol stimulated cAMP secretion in both astrocyte cultures and astrocyte-rich mixed cultures of astrocytes and neurons. However, no extracellular cAMP was detectable in neuron- enriched astrocyte-poor cultures. Extracellular adenosine increased in response to isoproterenol in the astrocyte-rich mixed cultures, but not in the neuron-enriched astrocyte-poor cultures. After 30 min exposure to isoproterenol, the concentration of adenosine in the extracellular medium increased by 47% in 56 experiments in the mixed astrocyte-rich cultures. In order to establish whether the adenosine that accumulates in response to isoproterenol stimulation actually derives from extracellular cAMP, phosphodiesterase inhibitors were tested for their ability to block isoproterenol-stimulated adenosine accumulation. Isobutylmethylxanthine (IBMX; 100 microM), RO 20–1724 (180 microM), carbazeran (10 microM), dipyridamole (10 microM), and trifluoperazine (10 microM) had no inhibitory effect on the isoproterenol-stimulated accumulation of extracellular adenosine. However 100 microM IBMX plus 180 microM RO 20–1724 effectively blocked isoproterenol-stimulated adenosine accumulation and, as expected, increased extracellular cAMP. As a further test of the origin of isoproterenol-stimulated adenosine accumulation, we attempted to block this phenomenon by blocking cAMP secretion itself. For this purpose probenecid, a known inhibitor of cAMP secretion in many different cell types, was used. We found that probenecid at 1 mM blocked isoproterenol-stimulated adenosine accumulation. These studies suggest that one potentially important source of extracellular adenosine in the cerebral cortex is endogenous extracellular cAMP, secreted from astrocytes in response to beta- adrenergic receptor stimulation. Since the receptors of neuromodulators other than norepinephrine may also be coupled to adenylyl cyclase in the cerebral cortex, there may be several neuromodulatory systems that regulate extracellular adenosine levels by this mechanism.