Abstract An image-splitter television microscope for measurement of microvascular dimension changes in the rat exteriorized mesentery was used to investigate the role of endothelium-derived relaxing factor (EDRF)/nitric oxide (NO) in the microcirculation. This was done by studying the effect of NG-monomethyl-L-arginine (L-NMMA), a specific inhibitor of NO formation, on the responses induced by acetylcholine (an endothelium-dependent vasodilator agent) and sodium nitroprusside (an endothelium-independent vasodilator agent). The effect of acetylcholine on mesenteric A 2 arterioles was impaired by previous application of L- but not D-NMMA to the preparations whereas the vasodilator response to sodium nitroprusside was not altered. The effect of L-NMMA was slow to disappear, unless accelerated by a 3-fold molar excess of L- but not D-arginine. It is suggested that EDRF/NO might be involved in the vasodilator response to endothelium-dependent agents such as acetylcholine at the microcirculatory level and that L-arginine might be the physiological precursor of NO.