In pentobarbital-anesthetized rats we investigated the role of the endothelium in the dilation of third-order arterioles of cremaster muscle to acetylcholine, adenosine, and sodium nitroprusside in vivo. Responses to the topical administration of these agents were measured with image shearing and recorded with video microscopy before and after light-dye (L-D) treatment of a 50- to 100-microns segment of the arteriole under study. L-D treatment consisted of intravascular administration of sodium fluorescein and the illumination of a discrete area of the arteriole under study with its excitation light from a mercury lamp. Before L-D treatment, acetylcholine (10(-7) to 10(-5) M) and adenosine (10(-6) to 10(-4) M) produced dose-dependent increases in arteriolar diameter (vasodilation). After L-D treatment of the arteriolar segment, administration of 10(-7) M acetylcholine evoked a vasoconstriction (-19% from control), and the dilator responses to 10(-6) and 10(-5) M were inhibited by 91 and 77%, respectively. In contrast, the arteriolar dilator responses to all doses of adenosine and sodium nitroprusside (2 x 10(-7) M) were not altered by this treatment. In addition, the dilator responses to acetylcholine were not changed in the nonilluminated, distal, and proximal segments of the arterioles. These findings suggest that L-D treatment selectively alters the function of the endothelium resulting in the loss of vasodilation to acetylcholine, whereas arteriolar smooth muscle function does not appear to be affected.