The involvement of endothelial adenosine A₂ receptor-cAMP system in A₂ receptor-mediated vasodilation in human aortic endothelial cells (HAEC) was investigated. Reverse transcription-polymerase chain reaction (RT-PCR) revealed the expression of both A_2a and A_2b receptors mRNA in HAEC. In HAEC, YT-146 (selective A₂ receptor-agonist) produced a dose dependent increase of cAMP production. This increase was inhibited by theophylline. YT-146 also showed a vasodilatory action in isolated rat aorta. The removal of endothelium significantly attenuated this vasodilatory effect. Our results provide the first evidence for the expression of both subtypes of the A_2a and A_2b receptors which regulate cAMP production in human endothelial cells. The present results also suggest that A₂ receptor-cAMP system was involved in the endothelium-dependent vasodilatory actions and may play important roles in regulating vascular functions of HAEC.