Experiments were undertaken in the mesentery of anesthetized cats and rabbits to investigate whether the endothelium of resistance vessels mediates relaxation of arteriolar smooth muscle through the endothelium-derived relaxing factor(s) (EDRF). The microcirculation was visualized by transillumination using intravital microscopy and a Millikan camera. Pictures were obtained at a rate of 400 frames/second. Arteriolar mean diameter and surface area were computer calculated. The animals were divided in two groups. In one group of experiments, EDRF was inactivated by direct injection of hydroquinone (HQ) into the superior mesenteric artery and the second group served as control. Norepinephrine or vasopressin constricted while acetylcholine or Ca-ionophore A23187 dilated arterioles. Hydroquinone failed to inhibit arteriolar dilation in situ. The effect of HQ on the endothelium in situ was ascertained by bioassay of superior mesenteric artery strips. Our results cast doubt on the role of EDRF in the dilation of mesenteric arterioles in felines and rabbits.