We investigated whether loss of myocardial protection after ischemic preconditioning (IP) is related to the extent of deactivation of activated ecto-5'-nucleotidase. The coronary arteries of mongrel dogs were occluded four times for 5 min separated by 5 min of reperfusion (IP). Five (IP1), 30 (IP2), 60 (IP3), and 120 min (IP4) after the fourth 5-min coronary occlusion or after a corresponding nonischemic period (control groups), the coronary arteries were occluded for 90 min followed by 6 h of reperfusion. The infarct size-limited effect of IP gradually disappeared in the IP2 (21.6 +/- 3.9%) and IP3 (33.8 +/- 3.6%) groups compared with the IP1 group (8.3 +/- 1.6%) and returned to the control level in the IP4 group (39.9 +/- 5.2%). The increased ecto-5' -nucleotidase activity due to the IP procedure decreased according to the order of IP1 to IP4 groups. Infarct size was inversely correlated with ecto-5'-nucleotidase activity (P < 0.001). An inhibitor of ecto-5'-nucleotidase blunted the infarct size-limiting effect of IP. The infarct size-limiting effect of IP decreased as the activation of ecto-5'-nucleotidase was blunted. These results suggest that ecto-5'-nucleotidase activity plays a key role in the cardioprotection of IP.