Adenosine (Ado) plays an important role in regulation of coronary vascular tone with nitric oxide (NO) and ATP-sensitive K⁺ (K⁺_ATP) channels. In vitro, it was reported that subendocardial (Endo) arterioles are more sensitive to Ado than subepicardial (Epi) arterioles. The purpose of this study was to observe enhanced vasodilation of Endo arterioles directly and to evaluate possible roles of K⁺_ATP channels and NO in the different responses of Endo and Epi arterioles to Ado-induced vasodilation. We evaluated dilation of Endo and Epi arterioles (<120 µm) of beating canine hearts (n = 19) by Ado (20 and 50 µg · kg¹ · min¹ ic) before and after K⁺_ATP channel blockade (glibenclamide; 200 µg/kg ic), inhibition of NO synthase [N^G-nitro-L-arginine methyl ester (L-NAME); 30 µg · kg¹ · min¹, 20 min ic], or glibenclamide + L-NAME using a novel needle-probe CCD intravital microscope. Ado induced dose-dependent vasodilation in both Epi and Endo arterioles, but vasodilation was greater in Endo arterioles, i.e., increase at 120 s (maximum dilation) after Ado (50 µg · kg¹ · min¹) was 17% in Endo and 13% in Epi arterioles (P < 0.01). Endo arteriole dilation was attenuated by blockade of K⁺_ATP channels from 18% (Ado) to 9% (Ado+glibenclamide) increase (P < 0.001) and by inhibition of NO synthase from 17% (Ado) to 9% (Ado+L-NAME) (P < 0.005). Epi arteriole vasodilation was attenuated by blockade of K⁺_ATP channels from 15 to 9% (P < 0.005) and inhibition of NO from 16 to 10% (P < 0.005). Suppression of vascular response was additive (Endo, 14 to 1%; Epi, 12 to 3%) with glibenclamide + L-NAME. We conclude that 1) the degree of Ado-induced vasodilation was greater in Endo than in Epi arterioles, with higher sensitivity of smaller arterioles in both layers and 2) transmural difference of arteriolar sensitivity to adenosine was abolished or reversed by K⁺_ATP channel blockade and/or by NO synthase inhibition, indicating crucial involvement of K⁺_ATP and NO in transmural sensitivity difference.