Objective: The aim was to test the hypotheses that acadesine (1) augments endogenous interstitial fluid (ISF) adenosine during ischaemia, and (2) reduces infarct size by adenosine receptor mediated mechanisms. Methods: To test these hypotheses, the left coronary artery of anaesthetised rabbits (n = 33) was occluded for 30 min and reperfused for 120 min. Acadesine (1 mg-kg⁻¹·min⁻¹ for 5 min, then 0.2 mg·kg⁻¹·min⁻¹) was infused intravenously beginning 30 min before coronary occlusion and ending 30 min after reperfusion. The area at risk was comparable in all groups, averaging 34.7(SEM 2.2%) of the left ventricle. In separate studies (n = 22), estimates of ISF adenosine and adenosine metabolites were obtained by cardiac microdialysis. Although dialysate adenosine levels increased significantly in the area at risk during ischaemia in the untreated group [from 0.044(0.008) to 0.339(0.146) μM], acadesine did not significantly augment dialysate adenosine levels before or during ischaemia [preischaemia = 0.094(0.032) μM; ischaemia = 0.542(0.262) μM]. In addition, there was no significant difference in dialysate adenosine concentrations during the first 10 min of reperfusion, after which adenosine levels returned to baseline levels. A 2.5-fold large dose failed to increase interstitial fluid adenosine. However, the adenosine receptor blocker 8-p-sulphophenyltheophylline (SPT) in the presence of acadesine increased ISF adenosine fourfold. Acadesine significantly (P < 0.05) reduced infarct size [n = 8, 19.7(2.9)% of risk area] compared with the untreated group [n = 8, 29.4(1.3)%]. This infarct size reduction with acadesine was antagonised by SPT given during ischaemia-reperfusion [n = 8, 46.2(3.0)%] or only during reperfusion [n = 9, 42.7(2.6)%. Conclusions: Acadesine reduces infarct size by an adenosine mediated mechanism, but this cardioprotective action is not associated with significantly augmented interstitial fluid adenosine levels.