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.