Heart failure after myocardial infarction (MI) is associated with endothelial dysfunction. There is conflicting evidence on the exact nature of this endothelial dysfunction and how endothelium-dependent vasodilation is affected by angiotensin-converting enzyme inhibitor (ACE-I) therapy. Furthermore, consequences of acute ACE-I withdrawal are largely unknown. Therefore, we studied the contribution of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) to the effects of ACE-I therapy and its withdrawal on endothelial function in MI rats. Rats were subjected to coronary ligation to induce MI and were assigned to quinapril or vehicle from 2 weeks to 8 months post-MI. In parallel, MI rats treated for 14 months with quinapril were subjected to treatment withdrawal for 0, 4, and 6 weeks. Acetylcholine (ACh)-induced relaxation and underlying endothelium-derived mediators were studied in isolated aortic rings. Long-term quinapril (8 months) resulted in markedly improved endothelium-dependent vasodilation in rats with myocardial infarction, which could be attributed to marked improvement in non-NO/prostanoid-mediated relaxation (ie, EDHF). After 14 months of follow-up, maximum vasodilation was still preserved by quinapril. Withdrawal after 14 months of treatment caused significantly impaired ACh-induced EDHF-mediated relaxation within 4 weeks. A marked reduction in EDHF-mediated relaxation caused this impairment. NO-mediated relaxation was unaffected. These findings highlight the importance of EDHF impairment in development of endothelial dysfunction after myocardial infarction and the possibility of improving EDHF-mediated vasodilation with chronic ACE inhibitor therapy. In addition, withdrawal of chronic ACE inhibition after MI should be considered carefully, as profound endothelial dysfunction may develop rapidly.