This study tested the hypothesis that cardiac ecto-5′-nucleotidase (ecto-5′-NT) activity during ischemic preconditioning (PC) contributes to augmented tolerance against ischemia, thereby reducing infarct size in the rabbit heart in situ. The effects of α,β-methylene-adenosine diphosphate (AOPCP), a selective inhibitor of ecto-5′-NT, on cardiovascular responses to AMP were measured to establish in vivo activities of the enzyme and its inhibitor. Left atrial infusion of AOPCP (0.75 mg ⋅ kg⁻¹ ⋅ min⁻¹) raised AOPCP plasma levels to 138 μM; under these conditions negative chronotropic and inotropic effects of AMP were blocked, demonstrating essentially full inhibition of ecto-5′-NT in the heart in situ. This AOPCP-blocked heart in situ model was used to examine the proposed contribution of ecto-5′-NT in ischemic PC. Myocardial infarction caused by 30-min ischemia was followed by 3-h reperfusion. Infarct size (IS) was measured and expressed as a percentage of the size of the area at risk (%IS/AR). In untreated controls, %IS/AR was 38.1 ± 3.8%; PC (5-min ischemia, 5-min reperfusion) markedly reduced %IS/AR to 10.0 ± 2.0%. Essentially identical IS reductions by PC were observed in AOPCP-blocked animals (%IS/AR = 13.8 ± 2.2 and 13.3 ± 1.8% in rabbits receiving AOPCP at 0.75 and 1.50 mg ⋅ kg⁻¹ ⋅ min⁻¹, respectively); here plasma AOPCP levels were established before and during PC but not during the subsequent prolonged ischemia. As expected, AOPCP also did not affect %IS/AR in non-PC controls (%IS/AR = 35.5 ± 3.7%). In contrast but as predicted, adenosine-receptor blockade by 8-phenyltheophylline (10 mg/kg iv) substantially attenuated IS reduction by PC in both AOPCP-blocked and control hearts (%IS/AR = 25.2 ± 4.3 and 21.8 ± 2.2%, respectively;P < 0.05 vs. PC alone). The results demonstrate that cardiac ecto-5′-NT is not required for ischemic PC against infarction in the rabbit.