Nitroxyl (HNO), a potential heart failure therapeutic, is known to target cysteine residues to form sulfinamides and/or disulfides. Because HNO-derived modifications may depend on their local environment, we have investigated the reactivity of HNO with cysteine derivatives and C-terminal cysteine-containing peptides at physiological pH and temperature. Our findings indicate that the nature of HNO-derived modifications of C-terminal cysteines is affected by the C-terminal carboxylate. Apart from the lack of sulfinamide formation, these studies have revealed the presence of new products, a sulfohydroxamic acid derivative (RS(O)2NHOH) and a thiosulfonate (RS(O)2SR), presumably produced under our experimental conditions via the intermediacy of a cyclic structure that is hydrolyzed to give a sulfenic acid (RSOH). Moreover, these modifications are formed independent of oxygen.