We have investigated the role of adenosine and its analogs on vasorelaxation of mouse aorta in intact endothelium with rank order of potency as follows: 5′-N-ethylcarboxamidoadenosine (NECA) > 2-chloroadenosine > adenosine ≫ CGS-21680, which is consistent with the profile of A_2B-adenosine receptor (A_2BAR). In endothelium-intact tissues, acetylcholine produced relaxation ranging from 65 to 80% in phenylephrine (PE, 10⁻⁷ M)-precontracted mouse aorta, whereas no relaxation was observed in endothelium-denuded tissues. The A_2BAR antagonist alloxazine (10⁻⁵ M) shifted concentration-response curve for NECA (EC₅₀ = 0.005 × 10⁻⁵ M) to the right with an EC₅₀ of 2.8 × 10⁻⁵ M, demonstrating that this relaxation is partially dependent on functional endothelium mediated predominantly via A_2BAR in this tissue. This conclusion was further supported by the following findings: 1) in the endothelium-intact mouse aorta, the EC₅₀ values for NECA and adenosine were found to be 0.05 and 1.99 × 10⁻⁴ M, respectively; however, in denuded endothelium, these values were 0.098 and 3.55 × 10⁻⁴ M, respectively; 2) NECA-induced relaxation was significantly blocked by N^G-nitro-l-arginine methyl ester (l-NAME; 10⁻⁴ M) in endothelium-intact tissues, which was reversed by pretreatment with l-arginine (10⁻⁴ M), whereas no significant inhibition was found in endothelium-denuded tissues; 3) total nitrites and nitrates (NOx) in intact endothelium with l-NAME (10⁻⁴ M) alone and in combination with l-arginine were 59% (P < 0.05) and 96%, respectively, in comparison with control (PE + NECA); and 4) endothelial nitric oxide synthase gene expression was found to be 67% (P < 0.05) less in endothelium-denuded as opposed to endothelium-intact mouse aorta. Thus these data demonstrate that adenosine-mediated vasorelaxation is partially dependent on A_2BAR in mouse aorta.