Experiments were designed to test whether preconditioning protects against chronic endothelial injury after ischemia and reperfusion. Coronary arteries were isolated from rats subjected to sham surgery or 20 min of ischemia followed by 1 h, 1 day, 1 wk, or 1 mo of reperfusion without or with preconditioning. The endothelium-dependent relaxations to acetylcholine (ACh; assessed in vitro) were markedly reduced after ischemia and 1 h of reperfusion (31 +/- 6 vs. 57 +/- 6% in sham; P < 0.01) and did not recover after longer durations of reperfusion (1 mo: 32 +/- 5 vs. 56 +/- 2%; P < 0.01). The impaired response to ACh was restored by preconditioning at all time points (1 h: 53 +/- 6; 1 mo: 65 +/- 4%). After 1 mo, the potency of ACh in preconditioned arteries was also increased compared with that in sham animals. Electron microscopy showed marked endothelial damage after 1 h of reperfusion and signs of regenerated endothelium after 1 mo of reperfusion. Both acute and chronic ultrastructural changes were prevented by preconditioning. Thus preconditioning, in addition to protecting myocardial cells, also protects against chronic reperfusion-induced endothelial injury, both in terms of functional and structural changes.