Current cellular models of ischemic preconditioning (IPC) rely on inducing preconditioning in vitro and may not accurately represent the complex pathways triggered by IPC in the intact heart. Here, we show that it is possible to precondition the intact heart and to subsequently isolate individual ventricular myocytes that retain the protection triggered by IPC. Myocytes isolated from Langendorff perfused hearts preconditioned with 3 cycles of ischemia/reperfusion were exposed to metabolic inhibition and re-energization. Injury was assessed from induction of hypercontracture, loss of Ca²⁺-homeostasis and of contractile function. IPC induced an immediate window of protection in isolated myocytes, with 64.3 ± 7.6% of IPC myocytes recovering Ca²⁺-homeostasis compared to 16.9 ± 2.4 % of control myocytes (p<0.01). Similarly, 64.1 ± 5.9 % of IPC myocytes recovered contractile function compared with 15.3 ± 2.2 % of control myocytes (p<0.01). Protection was prevented by the presence of 0.5mM 5-hydroxydeanoate during the preconditioning stimulus. This early protection disappeared after 6 hours but a second window of protection developed 24 hours after preconditioning, with 54.9 ± 4.7 % of preconditioned myocytes recovering Ca²⁺-homeostasis compared to 12.6 ± 2.9 % of control myocytes (p<0.01). These data show that "true" IPC of the heart confers both windows of protection in the isolated myocytes, with a similar temporal relationship to in-vivo preconditioning of the whole heart, mediated in part by a reduction in Ca²⁺-loading. The model should allow future studies in isolated cells of the protective mechanisms induced by true ischemia.