Little is known of the regulation of skeletal muscle microvascular exchange under resting or stimulating conditions. Adenosine (ADO) levels in skeletal muscle increase during physiological (exercise) and pathological (hypoxia, inflammation, and ischemia) conditions. Later stages of these pathologies are characterized by the loss of vascular barrier integrity. This study focused on determining which ADO receptor mediates the robust reduction in microvessel permeability to rat serum albumin (P_s^RSA) observed in juvenile female rats. In microvessels isolated from abdominal skeletal muscle ADO suffusion induced a concentration-dependent reduction in arteriolar [log (IC₅₀) = -9.8±0.2 M] and venular [log (IC₅₀) = -8.4±0.2M] P_s^RSA. RT-PCR and immunoblot analysis demonstrated mRNA and protein expression of ADO A₁, A_2A, A_2B, and A₃ receptors in both vessel types and immunofluorescence assay revealed expression of the four subtype receptors in the microvascular walls (endothelium and smooth muscle). P_s^RSA responses of arterioles and venules to ADO were blocked by 8-SPT, a non-selective A₁ and A₂ antagonist. An A_2A agonist, CGS21680, was more potent than the A₁ agonist, CPA, or most selective A_2B agonist, NECA. The ability of CGS21680 or ADO to reduce P_s^RSA was abolished by A_2A antagonist, ZM241385. An adenylyl cyclase inhibitor, SQ22536, blocked P_s response to ADO. In aggregate, these results demonstrate that in juvenile females (prior to the production of the reproductive hormones), ADO enhances skeletal muscle arteriole and venule barrier function predominantly via A_2A receptors using activation of adenylyl cyclase signaling mechanisms.