OBJECTIVE Adenosine is a cAMP-elevating vasodilator that induces both endothelium-dependent and -independent vasorelaxation. An increase in cytosolic Ca(2+) ([Ca(2+)](i)) is a crucial early signal in the endothelium-dependent relaxation elicited by adenosine. This study explored the molecular identity of channels that mediate adenosine-induced Ca(2+) influx in vascular endothelial cells. METHODS AND RESULTS Adenosine-induced Ca(2+) influx was markedly reduced by L-cis-diltiazem and LY-83583, two selective inhibitors for cyclic nucleotide-gated (CNG) channels, in H5V endothelial cells and primary cultured bovine aortic endothelial cells (BAECs). The Ca(2+) influx was also inhibited by 2 adenylyl cyclase inhibitors MDL-12330A and SQ-22536, and by 2 A(2B) receptor inhibitors MRS-1754 and 8-SPT, but not by an A(2A) receptor inhibitor SCH-58261 or a guanylyl cyclase inhibitor ODQ. Patch clamp experiments recorded an adenosine-induced current that could be inhibited by L-cis-diltiazem and LY-83583. A CNGA2-specific siRNA markedly decreased the Ca(2+) influx and the cation current in H5V cells. Furthermore, L-cis-diltiazem inhibited the endothelial Ca(2+) influx in mouse aortic strips, and it also reduced 5-N-ethylcarboxamidoadenosine (NECA, an A(2) adenosine receptor agonist)-induced vasorelaxation. CONCLUSIONS CNGA2 channels play a key role in adenosine-induced endothelial Ca(2+) influx and vasorelaxation. It is likely that adenosine acts through A(2B) receptors and adenylyl cyclases to stimulate CNGA2.