We have previously reported that adenosine formed in response to reduced arteriolar and/or tissue PO₂ preserves endothelial nitric oxide (NO) synthesis during sympathetic vasoconstriction in the rat intestine. To more precisely identify the site and mechanism of adenosine formation under these conditions, we tested the hypothesis that ATP released in response to reduced O₂ levels serves as a source of adenosine. Direct application of ATP to the wall of first-order arterioles elicited dose-dependent dilations of 15-33% above resting diameter that were reduced by 71-80% by the 5'-ectonucleotidase inhibitor ,-methyleneadenosine 5'-diphosphate (AOPCP, 4.5 × 10⁵ M) and completely abolished by N^G-monomethyl-L-arginine (L-NMMA, 10⁴ M). Under control conditions, sympathetic nerve stimulation at 3 and 8 Hz induced arteriolar constrictions of 11 ± 1 and 19 ± 1 µm, respectively. These responses were enhanced by 58-69% in the presence of L-NMMA or when local PO₂ was maintained at resting levels. However, in the presence of AOPCP, the enhancing effects of L-NMMA and the high O₂ superfusate on sympathetic constriction were preserved. These results suggest that, although exogenously applied ATP can stimulate arteriolar NO release in the intestine largely through its sequential extracellular hydrolysis to adenosine, this process does not contribute to adenosine formation and sustained NO release during sympathetic constriction in this vascular bed.