The aims of the current study were to examine the efflicacy of acetylcholine (ACh) to mimic ischemic preconditioning in dogs and to determine the role of nitric oxide (NO), muscarinic receptors, and ATP-sensitive K+ (KATP) channels in mediating its effects. Barbital-anesthetized open-chest dogs were subjected to 60 minutes of left anterior descending coronary artery (LAD) occlusion followed by 4 hours of reperfusion. Preconditioning was elicited by 10 minutes of LAD occlusion followed by 10 minutes of reperfusion before the 60-minute occlusion period. ACh (3 or 10 gg/min) or an equivalent volume of saline was infused into the LAD for 10 minutes, followed by a 10-minute drug-free period before the 60-minute ischemic insult. In other groups, the specific NO synthesis inhibitor NG -monomethyl-l-arginine (L-NMMA, 4 mg/min), the muscarinic receptor antagonist-NO synthesis inhibitor nitro-l-arginine methyl ester (L-NAME, 3 mg/min), or the specific KATP channel blocker 5-hydroxydecanoate (5-HD, 3 mg/min) was infused with ACh into the LAD for 10 minutes. The infusion of L-NAME, L-NMMA, or 5-HD was started 2 minutes before ACh infusion. Transmural myocardial blood flow was measured at 5 minutes of occlusion, and infarct size was determined by triphenyltetrazolium staining and expressed as a percentage of the area at risk. There were no significant differences in collateral blood flow or the area at risk between groups. Preconditioning produced a marked reduction (P<.05) in infarct size (6.2±3.0% versus 26.1 ±5.7% in the control group). Two doses of ACh (3 and 10 μg/min) also resulted in a marked dose-dependent decrease in infarct size (14.4±5.0% and 10.6±3.1%, respectively) similar to that observed with preconditioning. Simultaneous treatment with either L-NAME or 5-HD completely abolished the protective effects of the high dose (10 μg/min) of ACh (25.6±3.1% and 27.0±3.3%, respectively), whereas treatment with L-NMMA had no effect on ACh-induced infarct size reduction. These results indicate that ACh mimics ischemic preconditioning in dogs and provide evidence that the protection may be mediated via myocardial muscarinic receptors and the cardiac KATP channel but not through an NO-related mechanism. These data also indicate that interpreting results obtained using L-NAME as a specific NO synthesis inhibitor in the presence of a muscarinic agonist should be performed with caution.