Nitroxyl (NO-) is the one-electron reduction product of nitric oxide (NO.). Recently, NO- generating compounds were shown to possess potent vasorelaxant activity and this was attributed to the ready conversion of NO- to NO.. Because of its metastable character, direct chemical detection of NO- or its conjugated acid, HNO, has not been accomplished yet. In order to gain further insight into the cellular mode of action of NO- generating compounds we aimed at finding a means to discriminate NO- from NO. by bioassay. Using isolated rat aortic rings in organ baths, we here show that high concentrations of L-cysteine cause complete inhibition of the vasorelaxant response to NO- (generated from Angeli's salt and sodium nitroxyl) whereas responses to authentic NO. and S-nitrosocysteine are largely enhanced. Preliminary results indicate that the inhibition by L-cysteine of NO- activity may be mediated in part by enzymatic and non-enzymatic mechanisms. Whether or not NO- generating compounds will have promising therapeutic potential as a new class of NO.- donors will not least depend on their interference with enzymatic routes susceptible to inhibition by NO-.