Diabetes mellitus (DM) blocks protection by ischaemic preconditioning (IPC) by an unknown mechanism. We investigated the effect of IPC on MAPKs and HSP27 activation in diabetic and non-diabetic rat hearts in vivo. Two groups of anaesthetized non-diabetic (IPC) and diabetic (DM+IPC) rats underwent a preconditioning protocol (3 cycles of 3-min coronary occlusion and 5 min reperfusion). Two further groups served as untreated controls (CON and DM). Then hearts were excised for protein measurements by western blot. Instead of heart excision, four additional groups underwent 25 min coronary occlusion followed by 2 h reperfusion to induce myocardial infarction. In these animals infarct size (IS) was measured. IPC reduced infarct size in the non-diabetic rats from 55±21% to 31±13% (mean±SD, P<0.05) but not in the diabetic animals (DM: 52±12%, DM+IPC: 55±9%, P=1.0). In diabetic rats, IPC induced phosphorylation of p38 (0.58±0.40 vs. 0.24±0.15 AVI) ERK-1 (1.21±0.80 vs. 0.76±0.52 AVI), ERK-2 (0.83±0.52 vs. 0.50±0.41 AVI) and HSP27 (0.79±0.41 vs. 0.35±0.22 AVI, each P<0.05). Activation of these proteins was also observed in the non-diabetic animals (P<0.05 except for ERK-1 and HSP27). We conclude that protection by IPC is blocked by DM in the rat heart in vivo without affecting MAPK and HSP27 activation.