The present study investigates infarct-reducing effects of blocking ischemia-induced opening of connexin43 hemichannels using peptides Gap19, Gap26 or Gap27. Cardioprotection by ischemic preconditioning (IPC) and Gap peptides was compared, and combined treatment was tested in isolated, perfused male rat hearts using function and infarct size after global ischemia, high-resolution respirometry of isolated mitochondrial and peptide binding kinetics as endpoints. The Gap peptides reduced infarct size significantly when given prior to ischemia plus at reperfusion (Gap19 76.2 ± 2.7, Gap26 72.9 ± 5.8 and Gap27 71.9 ± 5.8% of untreated control infarcts, mean ± SEM). Cardioprotection was lost when Gap26, but not Gap27 or Gap19, was combined with triggering IPC (IPC 73.4 ± 5.5, Gap19-IPC 60.9 ± 5.1, Gap26-IPC 109.6 ± 7.8, Gap27-IPC 56.3 ± 8.0% of untreated control infarct). Binding stability of peptide Gap26 to its specific extracellular loop sequence (EL2) of connexin43 was stronger than Gap27 to its corresponding loop EL1 (dissociation rate constant K_d 0.061 ± 0.004 vs. 0.0043 ± 0.0001 s⁻¹, mean ± SD). Mitochondria from IPC hearts showed slightly but significantly reduced respiratory control ratio (RCR). In vitro addition of Gap peptides did not significantly alter respiration. If transient hemichannel activity is part of the IPC triggering event, inhibition of IPC triggering stimuli might limit the use of cardioprotective Gap peptides.