THIRTEEN years after its discovery(1), there is still controversy over the chemical identity of endothelium-derived relaxing factor (EDRF). Although pharmacological and chemical evidence indicates that EDRF is nitric oxide(2), other candidates, including S-nitrosocysteine(3,4), complex(5), nitroxyl(6) and hydroxylamine(7), have been proposed to account for the vasorelaxant properties of EDRF. Such diverse compounds should differ in their stability and in reactivity with oxyhaemoglobin and with redox-active nucleophiles such as thiols. Here we use a bioassay to compare the pharmacodynamic profiles of these and other compounds with those of nitric oxide and EDRF. We find that some S-nitrosothiols, dinitrosyl-iron-cysteine complex, sodium nitroxyl and hydroxylamine can be eliminated as candidates as they are more stable than EDRF and less susceptible to inhibition by oxyhaemoglobin. Co-infusion of cysteine revealed major differences between the remaining candidates because it reduced the effect of authentic nitric oxide and EDRF on the bioassay tissues but enhanced the survival of S-nitrosocysteine and S-nitroso-cysteamine. Our results further support the evidence that EDRF, the pharmacological entity described by Furchgott and Zawadzki(1), is nitric oxide.