Diabetes mellitus (DM) and the resulting hyperglycaemia may interfere with the cardioprotective effect of ischaemic late preconditioning (LPC). Therefore, we investigated the effect of acute hyperglycaemia (part 1) and the effect of alloxan-induced DM with or without short-term insulin treatment (part 2) on LPC. Rabbits, chronically instrumented with a coronary artery occluder, were subjected to 30 min coronary artery occlusion and 2 h reperfusion (I/R) and infarct size (IS) was assessed. In part 1, four groups were studied. Controls were not treated further. LPC induced by a 5-min period of myocardial ischaemia 24 h before I/R reduced IS from 42-14 (controls) to 22-8% of the area at risk. Hyperglycaemia (600 mg dl^m1 by dextrose infusion, H₆₀₀) before and during the 30 min ischaemia tended to increase IS (57-16%, P=0.14 vs. controls) and blocked cardioprotection by LPC (H₆₀₀+LPC, 59-19%, P=1.0 vs. H₆₀₀, P=0.0003 vs. LPC). In part 2, LPC reduced infarct size from 43-13% (control) to 23-10% (P=0.003). In diabetic animals, IS was 39-11%, and cardioprotection by LPC could not be elicited (DM+LPC, 41-16%, P=0.02 vs. LPC). Short-term insulin treatment (I, 90 min before I/R, blood glucose <150 mg dl^m1) did not restore the cardioprotective effects of LPC (DM+I, 42-15%; DM+LPC+I, 40-10%, P=0.03 vs. LPC). It is concluded that acute hyperglycaemia and DM block the cardioprotection induced by LPC in rabbits and that the cardioprotection is not restored by short-term insulin treatment.