The vascular response to adenosine and its analogs is mediated by four adenosine receptors (ARs), namely, A1, A2A, A2B, and A3. A2AARs and/or A2BARs are involved in adenosine-mediated vascular relaxation of coronary and aortic beds. However, the role of A1ARs in the regulation of vascular tone is less well substantiated. The aim of this study was to determine the role of A1ARs in adenosine-mediated regulation of vascular tone. A1AR-knockout [A1AR(−/−)] mice and available pharmacological tools were used to elucidate the function of A1ARs and the impact of these receptors on the regulation of vascular tone. Isolated aortic rings from A1AR(−/−) and wild-type [A1AR(+/+)] mice were precontracted with phenylephrine, and concentration-response curves for adenosine and its analogs, 5′- N-ethyl-carboxamidoadenosine (NECA, nonselective), 2-chloro- N6-cyclopentyladenosine (CCPA, A1AR selective), 2-(2-carboxyethyl)phenethyl amino-5′- N-ethylcarboxamido-adenosine (CGS-21680, A2A selective), and 2-chloro- N6-3-iodobenzyladenosine-5′- N-methyluronamide (Cl-IBMECA, A3 selective) were obtained to determine relaxation. Adenosine and NECA (0.1 μM) caused small contractions of 13.9 ± 3.0 and 16.4 ± 6.4%, respectively, and CCPA at 0.1 and 1.0 μM caused contractions of 30.8 ± 4.3 and 28.1 ± 3.9%, respectively, in A1AR(+/+) rings. NECA- and CCPA-induced contractions were eliminated by 100 nM of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, selective A1AR antagonist). Adenosine, NECA, and CGS-21680 produced an increase in maximal relaxation in A1AR(−/−) compared with A1AR(+/+) rings, whereas Cl-IBMECA did not produce contraction in either A1AR(+/+) or A1AR(−/−) rings. CCPA-induced contraction at 1.0 μM was eliminated by the PLC inhibitor U-73122. These data suggest that activation of A1ARs causes contraction of vascular smooth muscle through PLC pathways and negatively modulates the vascular relaxation mediated by other adenosine receptor subtypes.