Pharmacological probes were used to assess the possible roles of guanosine 3',5'-cyclic monophosphate (cGMP)-associated endothelium-derived relaxing factor (EDRF) in mediating microvascular responses to endogenous and exogenous agents in vivo. Pentobarbital-anesthetized rats (Wistar, 6 wk old) were prepared for in vivo microscopic observation and quantification of changes in diameter of third-order arterioles (15-25 microns) in the cremaster muscle to topical application of all agents. In indomethacin-pretreated preparations, cremasteric arteriolar dilator responses to acetylcholine, bradykinin, or ATP, but not to adenosine, histamine, or prostaglandin E2, were inhibited by hydroquinone (50 microM). Vasodilation to acetylcholine was also inhibited by methylene blue (5 microM), a blocker of guanylate cyclase activation. Constrictor responses to norepinephrine were not affected by hydroquinone or methylene blue. The inhibition of acetylcholine-induced vasodilation by hydroquinone and methylene blue was reversed by superoxide dismutase, suggesting that superoxide anion antagonized the response. On the other hand, basal arteriolar diameters or responses to acetylcholine were not affected by oxygen metabolite scavengers. Unlike in isolated arteries, vasodilator responses to the calcium ionophore A23187 or arachidonic acid were completely antagonized by cyclooxygenase inhibition. These data suggest that EDRF could be involved in the control of microvascular tone; however, significant differences exist in the stimuli that elicit dilation through this mediator in small and large blood vessels.