Adenosine administration has been reported to lower blood pressure by activating specific membrane receptors. The rat and human heart and aorta have been previously found to express both A2-type adenosine receptors, which activate adenylyl cyclase, and A3 adenosine receptors (A3AR), which inhibit adenylyl cyclase. In the current study, we used A3 adenosine receptor (A3AR) knock-out mice to examine the hypothesis that the relative levels of the A2-type adenosine receptors and A3AR determine the steady-state levels of cAMP in the cells and may affect blood pressure. We found that the A3AR knock-out mice express normal levels of the A1- and A2-type adenosine receptors. In situ hybridization demonstrated that the level of A3AR is high in the vascular smooth muscle layer of aortas derived from wild-type mice, but is not detectable in the knock-out mice. The steady-state level of cAMP is elevated in the aorta and heart of knock-out mice, as compared to wild-type mice, but is not altered in platelets, where A3AR is not expressed naturally. A3AR knock-out mice possess a blood pressure comparable to this in wild-type mice. However, when challenged with adenosine, the knock-out mice display a further increase in cAMP levels in the heart and vascular smooth muscle and a significant decrease in blood pressure, as compared to wild-type mice. In contrast, the effect of adenosine on ADP-induced platelet aggregation is similar in both types of mice. These studies indicate that the A3AR affects the steady-state level of cAMP in the tissues where it is naturally expressed, and that it influences the blood pressure in response to adenosine.