Abstract In this study we investigated whether endothelial cells are involved in the dilation of third-order arterioles (14 to 22 μm) in response to adenosine triphosphate (ATP) in cremaster muscle of pentobarbital-anesthetized rats. Two light/dye ( L D ) techniques were employed to achieve selective, local endothelial impairment. One of these techniques utilizes a mercury lamp and sodium fluorescein, the other a Helium-Neon laser and Evans blue dye. L D treatment (illumination with the appropriate wavelengths of light in the presence of an intravascular dye) of a 20-to 100-μm segment of an arteriole resulted in a complete loss of arteriolar dilation in response to topical administration of acetylcholine (10 −6 M) and arachidonic acid (AA, 10 −5 M). These agents were applied in 100-μl aliquots without interrupting the continuous suffusion with Ringer-gelatin solution and caused a ∼70% increase in vascular diameter before the L D intervention. Selectivity of the impairment was assessed by arteriolar responses to the nonendothelium-dependent dilator agents adenosine (10 −5 M) and sodium nitroprusside (2 × 10 −7 M), which elicited the same degree of dilation before and after L D treatment. Under control conditions ATP (10 −6, 10 −5, and 10 −4 M) elicited dose-dependent increases in arteriolar diameter (from 38 to 74%). After impairment of arteriolar endothelium, dilation in response to all doses of ATP was significantly reduced. Theophylline (30 μ M) significantly inhibited arteriolar dilation in response to adenosine (10 −6, 10 −5, and 10 −4 M) but did not affect the responses to various doses of ATP. Moreover, impairment of endothelium enhanced constrictor responses of arterioles to norepinephrine (0.6 × 10 −8 M). These results indicate that arteriolar endothelium of skeletal muscle can mediate or modulate arteriolar responses to various vasoactive agents, suggesting that it has an important role in the regulation of blood flow.