The potency of adenosine and inosine as agonists at human adenosine receptors was examined in a functional assay using changes in cyclic AMP (cAMP) formation in intact Chinese hamster ovary (CHO) cells stably transfected with the human A1, A2A, A2B, and A3 receptors. Adenosine increased cAMP formation in cells expressing the A2A (EC(50): 0.7 microM) and A2B (EC(50): 24 microM) receptors and inhibited forskolin (0.3-3 microM)-stimulated cAMP formation in cells expressing the A1 (EC(50): 0.31 microM) and A3 receptors (EC(50): 0.29 microM). The potency of adenosine at the A2A and A2B receptors was not altered by the presence of the uptake inhibitor nitrobenzylthioinosine (NBMPR), whereas it was increased about 6-fold by NBMPR at the A1 and A3 receptors. In the presence of NBMPR, inosine was a potent agonist (EC(50): 7 and 0.08 microM at the A1 and A3 receptors, respectively), but with low efficacy especially at the A3 receptors. No effect of inosine was seen at the A(2) receptors. Caffeine, theophylline, and paraxanthine shifted the dose-response curve for adenosine at the A1, A2A, and A2B receptors. These results indicate that adenosine is the endogenous agonist at all human adenosine receptors and that physiological levels of this nucleoside can activate A1, A2A, and A3 receptors on cells where they are abundantly expressed, whereas pathophysiological conditions are required to stimulate A2B receptors to produce cyclic AMP.