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To investigate whether ischaemic post-conditioning (IPoC) combined with i.v. infusion of the nitric oxide (NO) substrate L-arginine at the onset of reperfusion exerts cardioprotective effect that is superior to either treatment given separately.Twenty-six anesthetized pigs were subjected to coronary artery (left anterior descending artery, LAD) ligation for 40 min followed by 4 h reperfusion. The pigs were randomized into five different groups receiving either i.v. vehicle, i.v. L-arginine, IPoC 4 × 60 s together with i.v. vehicle or IPoC together with i.v. L-arginine and a group with IPoC 8 × 30 s. All infusions were started 10 min before reperfusion.The infarct size of the vehicle group was 82 ± 4% of the area at risk. L-Arginine alone (79 ± 8%), IPoC 4 × 60 s vehicle (86 ± 3%) or IPoC 8 × 30 s vehicle (94 ± 7%) did not affect infarct size. L-Arginine together with IPoC significantly reduced infarct size to 59 ± 4% (P < 0.01). Except for higher LAD flow during early reperfusion in the IPoC L-arginine group, haemodynamic parameters did not differ between the four main groups. Heart rate and rate pressure product were lower during ischaemia and reperfusion in the IPoC 8 × 30 s vehicle group. In comparison with the vehicle group, there were no changes in the expression of Akt, phosphorylated Akt Ser473, inducible NO synthase, endothelial NO synthase (eNOS) or phosphorylated eNOS Ser1177 in the ischaemic/reperfused myocardium.L-Arginine given systemically at the onset of reperfusion protects the pig heart against ischaemia and reperfusion injury only when combined with IPoC. These results indicate that the combination of the two treatment strategies exerts cardioprotection.