In the present study we investigated the molecular effects of temperature preconditioning (TP) in freshly isolated adult rat cardiac myocytes. TP consisted of incubating myocytes at 16°C for 2 min, followed by 37°C for 3 min, twice. Contraction of myocytes was synchronised by electrical field stimulation at 1 Hz. Recovery of contractile function was measured following simulated ischaemia/reperfusion injury, using 7 min of perfusion with substrate-free Tyrode solution containing cyanide (2 mM) and iodoacetic acid (1 mM), followed by reperfusion with Tyrode solution for 10 min. A significant increase in contractile function after TP (52%±6 n=225, 3) was observed compared to control myocytes (29%±5 n=229, 3) (p<0.05). This protection was found to positively correlate with Ca2+ handling ability when Ca2+ levels were measured following 10 min of reperfusion. TP myocytes (132±18 nM n=67, 3) was found to significantly lower free intracellular Ca2+ concentration compared to control myocytes (301±53 nM n=69, 3) (p<0.005). In a cellular model of reperfusion injury TP was found to delay mitochondrial permeability transition pore (MPTP) opening (317±32 s n=49, 3) compared to controls (193±15 s n=44, 3) (p<0.0005). This was determined by loading myocytes with tetramethylrhodamine methyl ester (5 μM) followed by photodamage. Western blot analysis showed a significant increase of phospho- Erk in response to TP. In addition, Erk inhibitors (either U0126 (0.5 μM) or PD98059 (10 μM)) present during the TP protocol were found to abolish the TP-induced delay in MPTP opening. In conclusion, TP improved contractile function, Ca2+ handling and delayed MPTP opening. Data are presented as mean and SEM. n=myocytes, animals.