Previous work in our laboratory led us to postulate that N2a cells release adenosine into growth medium, where it acts at the extracellular adenosine receptors to modulate the sensitivity of the cells to the cyclic AMP-elevating effect of adenosine [Green, RD, J Pharmacol Exp Ther 201:610, 1977]. We have now devised a high-performance liquid chromatographic (HPLC) procedure capable of quantitating the concentrations of adenosine in cells and tissue culture media. Growth media of N2a cells and a variant of N2a cells deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT-) contain 10-20 nM adenosine, while that of a variant deficient in adenosine kinase (AK-) is elevated severalfold. It appears that the concentration of adenosine in growth media is determined by both the rate at which it is released by cells into the medium and the rate at which it is metabolized by adenosine deaminase present in the serum in the growth medium. Both N2a and AK- cells release considerable amounts of adenosine into serum-free medium (SFM) over a short period. Adenosine release is greater from AK- cells and is accelerated by erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA), a potent adenosine deaminase inhibitor. This accelerated release is retarded by dipyridamole and homocysteine. Surprisingly, dipyridamole and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20 1724), a potent phosphodiesterase inhibitor, stimulate basal adenosine release from N2a but not from AK- cells. It remains to be determined if this is due to an effect of these compounds on adenosine kinase. These results give further support for the hypothesis that adenosine in growth medium modulates the sensitivity of the cells to the cyclic AMP-elevating affect of adenosine, and furthermore they suggest that adenosine in growth media may tonically stimulate adenylate cyclase and affect processes controlled by the cyclic AMP:cyclic AMP-dependent protein kinase system.