Although previous studies suggested that the protection of late preconditioning (PC) against myocardial stunning is mediated by nitric oxide (NO), direct evidence that exogenous administration of NO attenuates myocardial stunning is lacking. Furthermore, although exogenous NO administration was shown to elicit a late PC phase, it is unknown whether NO donors also induce an early PC phase. Therefore, conscious rabbits underwent two experimental stages (3 days of six 4-min occlusion/4-min reperfusion cycles each) 2 wk apart. In study I, both stages were control stages (n = 7). In studies II and III, stage I was the control stage. On day 1 of stage II, seven rabbits received infusion of nitroglycerin (NTG; 2 microg. kg(-1). min(-1) iv) during the ischemia-reperfusion sequence, starting 30 min before the 1st occlusion and ending 10 min after the 6th reperfusion (study II). Another seven rabbits received infusion of NTG (2 microg. kg(-1). min(-1) iv) for 1 h followed by a 30-min washout interval and then underwent six 4-min occlusion/4-min reperfusion cycles (study III). In the control stage of all three studies, recovery of wall thickening (WTh) after occlusion/reperfusion cycles was markedly enhanced on days 2 and 3 compared with day 1, indicating late PC. In study II, infusion of NTG during the occlusion/reperfusion cycles on day 1 resulted in significant and sustained enhancement in WTh recovery. A similar attenuation of stunning was observed in study IV in six rabbits given intravenous infusion of S-nitroso-N-acetylpenicillamine (SNAP) during occlusion/reperfusion cycles. The magnitude of the protection afforded by NTG and SNAP was comparable to that afforded by the late ischemic PC phase. In contrast, in study III infusion of NTG before occlusion/reperfusion cycles did not enhance WTh recovery, indicating that NTG failed to induce an early PC effect against stunning. This study demonstrates that administration of hemodynamically inactive doses of two unrelated NO donors alleviates myocardial stunning in conscious rabbits, providing direct evidence for a protective action of NO in this setting.