We tested the hypothesis that late preconditioning (PC) against myocardial infarction is triggered by the formation of nitric oxide (NO). Conscious rabbits underwent a 30-min coronary occlusion followed by 3 days of reperfusion. In group I (control group, n = 10), rabbits were not preconditioned, whereas in group II (n = 10), they were preconditioned 24 h earlier with a sequence of six 4-min occlusion/4-min reperfusion cycles. Myocardial infarct size (tetrazolium staining) was reduced by 50% by PC (28.6 +/- 3.2% of the risk region in group II vs. 56.9 +/- 5.9% in controls, P < 0.05). This reduction in cell death was associated with improved recovery of myocardial function [systolic thickening fraction (by sonomicrometry) at 3 days: 2.0 +/- 11.0% of baseline in group II vs. -20.0 +/- 2.8% in group I, P < 0.05]. Group III rabbits (n = 11) underwent the same protocol as group II except that the rabbits received the NO synthase inhibitor N omega-nitro-L-arginine (L-NNA, 13 mg/kg) before the PC ischemia. In these animals, infarct size did not differ significantly from that observed in control rabbits, indicating that L-NNA completely blocked the development of late PC against myocardial infarction. In group IV (n = 9), rabbits received L-NNA as in group III, but without the six occlusion-reperfusion cycles, and were subjected to the 30-min occlusion 24 h later. In this group, infarct size did not differ from that observed in controls, demonstrating that pretreatment with L-NNA, in itself, did not affect the extent of cell death. Taken together, these results indicate that, in the conscious rabbit, the development of late PC against myocardial infarction is triggered by the generation of NO during the PC ischemia. It is proposed that NO plays a key role in the delayed myocardial adaptation to ischemic stress.