The contribution of luminally released endothelium-derived relaxing factor (EDRF) transported with the flowing blood to the control of smooth muscle tone of downstream arterioles was studied. The dilatory response of arterioles in the spinotrapezius muscle of the rat to superfusion of acetylcholine (ACH) was investigated by intravital microscopy before and after blockade of blood flow by micropipette occlusion upstream of the site of observation. Vessels were studied without treatment, after topical application of indomethacin (INDO, 3 · 10-5 M) to inhibit prostanoid production and after local treatment with NG-nitro-larginine (L-NNA, 2.9 · 10-3 M) to suppress local production of EDRF. In untreated vessels and after INDO, responses to ACH were not significantly different in the presence or absence of blood flow. After L-NNA, the responses to ACH were reduced to about 50%, but were again similar with or without flow. These results indicate that luminally transported EDRF does not play a significant role in controlling arteriolar tone in small skeletal muscle arterioles.