1. The malate-aspartate shuttle (MAS) is the main pathway for balancing extra- and intramitochondrial glucose metabolism. Pre-ischaemic shutdown of the MAS by aminooxyacetate (AOA) mimics ischaemic preconditioning (IPC) in rat glucose-perfused hearts. The aim of the present study was to determine the effects of fatty acids (FA) on cardioprotection by pre-ischaemic inhibition of the MAS. 2. Isolated rat hearts were divided into four groups (control; pre-ischaemic AOA (0.2 mmol/L); IPC; and AOA + IPC) and were perfused with 11 mmol/L glucose, 3% bovine serum albumin plus 0, 0.4 or 1.2 mmol/L FA. The perfusion protocol included 30 min global no-flow ischaemia and 120 min reperfusion. Infarct size (IS), haemodynamic recovery, glucose oxidation and lactate release were evaluated in all four groups. 3. Pre-ischaemic AOA reduced the IS of the left ventricle in hearts perfused with 0, 0.4 and 1.2 mmol/L FA compared with that in control hearts (26 ± 2% vs 53 ± 4%, 29 ± 3% vs 53 ± 4% and 61 ± 4% vs 81 ± 3%, respectively; P < 0.01 for all). After 2 h reperfusion, AOA improved haemodynamic recovery in the absence (52 ± 2 vs 27 ± 3 mmHg in the AOA and control groups, respectively; P < 0.001) but not in the presence, of FA. Both IPC and AOA + IPC reduced IS and improved haemodynamic recovery regardless of FA levels. Postischaemic glucose oxidation was suppressed by FA and did not differ significantly between the different groups. 4. In conclusion, the reduction in IS induced by pre-ischaemic MAS shutdown is not compromised by physiological FA concentrations. Transient MAS shutdown may be involved in IPC, but is not sufficient on its own as the underlying mechanism for IPC.