The gaseous free radical nitric oxide (NO^˙) is an important regulator of a variety of biological functions and also has a role in the pathogenesis of cellular injury. It has been generally accepted that NO^˙ is solely generated in biological tissues by specific nitric oxide synthases, NOSs, which metabolize arginine to citrulline with the formation of NO^˙. We report that NO^˙ can also be generated in the ischaemic heart by direct reduction of nitrite to NO^˙ under the acidotic and highly reduced conditions that occur. This NO^˙ formation is not blocked by NOS inhibitors, and with long periods of ischaemia progressing to necrosis, this mechanism of NO^˙ formation predominates. We observe that enzyme-independent NO^˙ generation results in myocardial injury with a loss of contractile function. The existence of this enzyme independent mechanism of NO^˙ formation has important implications in our understanding of the pathogenesis and treatment of tissue injury.