1. Although pharmacological preconditioning (PPC) has emerged as an alternative to ischaemic preconditioning (IPC) in cardioprotection, the efficacy of PPC compared with IPC has not been investigated. Because IPC is mediated by complex signalling cascades arising from multiple triggers, we have hypothesized that combined PPC is necessary to mimic IPC. 2. Isolated and perfused rat hearts underwent IPC by three cycles of 5 min ischaemia and 5 min reperfusion before 30 min global ischaemia followed by 120 min reperfusion. Adenosine (30 micromol/L), diazoxide (50 micromol/L) and s-nitroso-N-acetylpenicillamine (SNAP; 50 micromol/L) were added for 25 min just before (pretreatment modality) or 45 min before (PPC modality) the index ischaemia. 3. Ischaemic preconditioning significantly improved isovolumic left ventricular (LV) function and reduced infarct size. Although pretreatment with adenosine, diazoxide or SNAP alone was capable of reducing infarct size, PPC with each drug alone or in a combination of two drugs except for diazoxide plus SNAP failed to reduce infarct size. In contrast, PPC in combination with adenosine, diazoxide and SNAP (triple combination PPC) conferred significant improvement of LV function and reduction of infarct size that was as effective as IPC. 4. Cardioprotection afforded by triple combination PPC was abolished by the Gi/o-protein inhibitor pertussis toxin, the mitochondiral KATP channel inhibitor 5-hydroxydecanoate or the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (carboxy-PTIO). 5. Protein kinase C (PKC)-epsilon in the particulate fraction was activated throughout preconditioning ischaemia and reperfusion. Although PKC-epsilon was activated during treatment with adenosine, diazoxide or SNAP alone, it was inactivated after washout. In contrast, PKC-epsilon remained activated after triple combination PPC. The PKC inhibitor chelerythrine abolished activation of PKC-epsilon and cardioprotection afforded by IPC and triple combination PPC. 6. These results demonstrate that combined PPC with a G-protein-coupled receptor agonist, a mitochondrial KATP channel opener and an NO donor is necessary to mimic IPC and such synergistic cardioprotection is associated with enhanced and sustained activation of PKC-epsilon.