OBJECTIVE Ischemic preconditioning (IP) has been shown to attenuate intracellular Na+ accumulation and Ca2+ overload during ischemia and reperfusion, both of which are closely related to the outcome of myocardial damage. We compared the effects of single- and four-cycle IP in Na+,K(+)-activated adenosine 5'-triphosphatase (Na+,K(+)-ATPase) and Ca(2+)-activated adenosine 5'-triphosphatase (Ca(2+)-ATPase) activities in in vivo rabbit hearts, correlating these differences to the quality of protection against subsequent ischemia. METHODS The morphological outcome was evaluated in in vivo rabbit hearts subjected to 30 min of coronary occlusion and reperfusion for 180 min by assessing the ratio of infarct volume to risk zone volume. The effects of single- and four-cycle preconditioning ischemia were then examined. Another set of in vivo rabbit hearts was subjected to the measurement of ATPase activities at the conclusion of final preconditioning ischemia and at 60 min after reperfusion following 30 min of ischemia. RESULTS The infarct volume was reduced by single-cycle IP to 38% of that in the control group. The four-cycle IP further reduced the infarct volume, which was 11% of that in the control group. Na+,K(+)-ATPase activity at 60 min after reperfusion in the four-cycle group was increased to 172% of that in the control group (10.8 micromol ADP/h/mg protein), whereas no difference was found in the single-cycle group. On the other hand, Ca(2+)-ATPase activity at the conclusion of IP was increased by single-cycle IP, the value being 255% of that in the control group (4.9 micromol ADP/h/mg protein). The four-cycle IP further increased the activity, and the value was 158% of that in the single-cycle group. CONCLUSIONS Since increases in Na+,K(+)-ATPase and Ca(2+)-ATPase activities contribute to the decrease in intracellular Ca2+ concentration, the enhancement of these activities by four-cycle IP may be involved in the additional protection.