In recent years, major advances have been made toward understanding the role of nitric oxide (NO) in the ischemic biology of the heart. It is now clear that NO, either endogenous or exogenous, represents one of the most important defenses against myocardial ischemia–reperfusion injury. The purpose of this review is to provide an update on the cardioprotective actions of NO, with particular emphasis on the function of the inducible isoform of NO synthase (iNOS) and on the role of mitochondria in NO-mediated protection. This essay underscores some of the more prominent areas of ischemic biology that relate to NO, such as ischemic preconditioning, pharmacological cardioprotection, and gene therapy. The hypothesis that the late phase of preconditioning is mediated by increased iNOS activity resulting in enhanced NO bioavailability, first proposed by our group, is now widely accepted and can be regarded as a proven hypothesis. Likewise, the burgeoning field of postconditioning may share such a requirement for NO. Various drugs (e.g. statins, ACE inhibitors, angiotensin-receptor blockers, etc.) also produce salubrious effects in experimental models of myocardial infarction via their enhancement of NO bioavailability. Thus, NO appears to be a common mediator of the protection afforded by a wide array of seemingly unrelated pharmacological and nonpharmacological interventions, underscoring its fundamental role as a ubiquitous defense of the heart against ischemia and reperfusion. This review challenges the conventional wisdom that iNOS is deleterious during myocardial ischemia–reperfusion and instead proposes the concept that iNOS, when expressed in cardiac myocytes, is a profoundly protective protein. We also emphasize the emerging importance of the mitochondrial actions of NO. Although the precise molecular events remain to be defined, we propose that NO interacts with components of the electron transport chain and/or the mitochondrial permeability transition pore to limit post-ischemic myocardial damage, and that this action potentially provides a fundamental molecular explanation for the mechanism of NO-mediated cardioprotection.