Reactive Oxygen Species (ROS) generated during ischemic and pharmacological pre- and postconditioning are important triggers of cardioprotection.1,2 We have previously shown that insulin is cardioprotective both as a continuous infusion (Ins 15′ rep)3 and when given in short bursts of 3x30s at the immediate onset of reperfusion (InsPost, unpublished data), and that this protection is mediated via PI3K-Akt-p70s6K. But whether ROS is involved in insulin mediated cardioprotection is largely unknown. In addition, there has been variable success in combining cardioprotective treatments to give additional cardioprotection.4,5 Our study therefore explores (1) the effect of ROS-scavenging on insulin cardioprotection, and (2) the effect of combining IPost with insulin therapy. We used Langendorff-perfused rat hearts subjected to 20 min stabilization, 30 min of regional ischemia and 120 min of reperfusion. The hearts were randomized to either control (Ctr), IPost, InsPost , Ins 15′ rep or Ins 15′ rep + IPost, with or without the presence of the ROS scavenger N-mercaptopropionylglycine (MPG, 1 mM or 10 mM). Infarct size was determined by tetrazolium staining, and expressed as percent of area at risk. Values are expressed as means ± S.E.M. compared by one-way ANOVA and Fischer’s post hoc test. Significance level was set to 0.05 and all groups were n≥6. Our results show that both IPost and insulin treatment is dependent on ROS signalling (IPost 34.9±3.1 vs. IPost+MPG 57.0±3.7, InsPost 33.1±7.8 vs. InsPost+MPG 56.0±6.8, and Ins 15′ rep 36.0±5.9 vs. Ins 15′ rep+MPG 63.4±6.2, all p<0.05). MPG had no effect on infarct size alone (Ctr 51.8±5.5 vs. MPG 59.7±3.3 ns.). Combining insulin treatment with IPost did not afford any additional cardioprotection but abrogated it instead (IPost+Ins 1′ Rep 41.6±7.5, IPost+Ins 5′ Rep 57.0±3.6 and IPost+Ins 15′ Rep 60.8±3.8 vs. Ctr 51.8±5.5 ns.) Scavenging free radical formation with MPG did not affect the infarct size regardless of dose (Ctr 51.8±5.5, MPG 1mM 59.7±3.3 and MPG 10 mM 66.4±5.2 vs. IPost+Ins 15′ Rep + MPG 1 mM 49.7±7.1 and IPost + Ins 15′ Rep + MPG 10 mM 52.3±5.3 ns.) In conclusion, our data suggest that cardioprotection from insulin is dependent on ROS-signalling. Surprisingly, combining IPost and Ins 15′ rep did not afford any additional cardioprotection, but abrogated it via blunted Akt-phosphorylation. The protection was not regained by ROS scavenging with MPG regardless of dose.