Elucidation of the subcellular mechanism of myocardial ischemic preconditioning should be facilitated by precise knowledge of the biology of the cardioprotective response. Any proposed molecular mechanism for preconditioning must be initiated during the required ischemic stress period. The studies reported in this paper were undertaken to determine whether the infarct-limiting effect of four 5-min episodes of ischemia interspersed by reperfusion can be achieved by a single 5-min episode. Adult open-chest mongrel dogs, premedicated with the analgesic butorphanol, and anesthetized with sodium pentobarbital, underwent occlusion of the circumflex coronary artery for 60 min, followed by reperfusion of 3 h. Treated dogs were preconditioned with one, two or four cycles of 5-min occlusion followed by reperfusion. Additional dogs, not premedicated with butorphanol, were either untreated (not preconditioned) or preconditioned with one cycle of ischemia. Infarcts were identified using triphenyl-tetrazolium chloride (TTC) macrochemistry and infarct size (as % of area-at-risk, AAR) was measured and analyzed (using analysis of covariance [ANCOVA]) with respect to coronary collateral blood flow (measured using radioactive microspheres). Four 5-min cycles of preconditioning ischemia markedly limited infarct size. Two cycles were as effective as four. In contrast, infarct size was not different from control infarct size after a single episode of preconditioning ischemia. However, when pentobarbital anesthesia was used without premedication with butorphanol, a single 5-min ischemic stress did induce cardioprotection. Thus, the ischemic stress required for myocardial preconditioning in dogs is dependent on the anesthetic and premedication protocol employed. A single 5-min stimulus is effective in dogs anesthetized with pentobarbital. Premedication with the opioid analgesic, butorphanol, increases the threshold for induction of cardioprotection.