Hydrogen sulfide (H(2)S) has been increasingly recognized as an important signaling molecule that regulates both blood pressure and neuronal activity. Attention has been drawn to its interactions with another gasotransmitter, nitric oxide (NO). Here, we provide evidence that the physiological effects observed upon the application of sodium nitroprusside (SNP) and H(2)S can be ascribed to the generation of nitroxyl (HNO), which is a direct product of the reaction between SNP and H(2)S, not a consequence of released NO subsequently reacting with H(2)S. Intracellular HNO formation has been confirmed, and the subsequent release of calcitonin gene-related peptide from a mouse heart has been demonstrated. Unlike with other thiols, SNP reacts with H(2)S in the same way as rhodanese, i.e., the cyanide transforms into a thiocyanate. These findings shed new light on how H(2)S is understood to interact with nitroprusside. Additionally, they offer a new and convenient pharmacological source of HNO for therapeutic purposes.