Controversy surrounds the involvement of nitric oxide (NO) in myocardial ischaemia-reperfusion injury and the balance between deleterious and beneficial effects. NO synthase (NOS) is expressed constitutively as two isoforms: endothelial (eNOS) and neuronal (nNOS). Knockout mice lacking the gene for either eNOS (eNOS KO) or nNOS (nNOS KO), were compared with wild-types (WT) during a protocol of global ischaemia-reperfusion injury. Thirty-six mouse hearts (12 from each group) were isolated and the aorta cannulated for Langendorff perfusion with modified Krebs solution at constant pressure. An apical suture connected the left ventricle to a force transducer via a light weight coupling rod. Following stabilization hearts were subjected to 30 min of global ischaemia at 37 degrees C. During 30 min reperfusion, the recovery of baseline force-rate product (F%) was recorded. Hearts were then stained with tetrazolium, frozen, sliced, and fixed with formalin. Slices were compressed between plexiglas plates, and a magnified video image digitized to allow planimetry for infarct size (as percentage of ventricular volume I/R). Although recovery of contractile function did not differ between groups, eNOS KOs suffered significantly larger infarcts than WT or nNOS KOs (41 v 33 and 30% respectively, P<0.05 for both comparisons). A protective role for eNOS against global ischaemia-reperfusion injury has been demonstrated for the first time in murine myocardium. This may have important clinical implications for future pharmacotherapy to enhance myocardial protection.