Intravital microscopy was used to study the effect of endothelium-derived relaxing factor (EDRF) on microvascular adrenoceptor sensitivity in rat cremaster skeletal muscle.NG-Monomethyl l-arginine (L-NMMA, 1–300 μM), an inhibitor of EDRF formation, produced concentration-dependent constriction of arterioles and venules. When an intermediate amount of α1- versus α2-adrenoceptor tone was first produced with bath-added norepinephrine (NE) in the presence of rauwolscine or prazosin, L-NMMA caused constriction with greater potency and efficacy during α2than during α1tone. During localized α1or α2constriction along an arteriole by perivascular micropipette suffusion of NE in the presence of rauwolscine or prazosin, again, bath-added L-NMMA produced constriction with greater potency during α2than during α1constriction. Like L-NMMA, disruption of EDRF release by microembolization caused baseline arteriole constriction and selectively increased α2sensitivity 75-fold. Although these findings support the hypothesis that endothelial cells possess α2-adrenoceptors that promote EDRF release, a greater susceptibility of α2than α1constriction to EDRF inhibition could also account for the results. In support of this latter possibility, α2constriction was approximately 50-fold more susceptible than α1constriction to inhibition by the EDRF-like nitrodilator nitroprusside. The similarity in magnitude of this difference in sensitivity with the difference obtained in the embolization experiments does not support the hypothesis that microvascular endothelial cells in skeletal muscle possess EDRF-promoting α2-adrenoceptors. However, these data do suggest that endogenous EDRF release modulates basal arteriole and venule tone and that α2-adrenoceptor constriction is more sensitive thana, constriction to inhibition by EDRF.