Pharmacological uncoupling of mitochondrial oxidation from phosphorylation promotes preconditioning-like cardioprotection in the isolated rat heart. We hypothesized that modest mitochondrial uncoupling may be a critical cellular event in orchestrating preconditioning. Human-derived Girardi cells and murine C2C12 skeletal myotubes were preconditioned using simulated ischemia, adenosine, and diazoxide. Cell viability after 6 hours of simulated ischemia was measured using lactate dehydrogenase release and propidium iodide uptake. Mitochondrial inner membrane potential (&Dgr;&PSgr;m) was investigated by flow cytometry, cellular ATP by recombinant firefly-luciferase bioluminescence, and cellular oxygen consumption using oximetry. Preconditioning enhanced cell viability with attenuation of lactate dehydrogenase release (≥30%,P<0.05 versus ischemic controls) and a reduction in propidium iodide uptake by ≥26% versus ischemic controls after simulated ischemia in both cell lines. In Girardi cells, preconditioning induced the following phenotype immediately before index ischemia: (1) decreased &Dgr;&PSgr;m (JC-1: simulated ischemia 90±3%, adenosine 82±7%, diazoxide 87±4%, versus control 100%,P<0.05); (2) attenuation in cellular ATP levels (CTL 0.21±0.03 nmol/L ATP/&mgr;g protein, simulated ischemia 0.12±0.02, adenosine 0.15±0.02, diazoxide 0.11±0.02,P<0.05); and (3) enhanced cellular oxygen consumption (control 2.3±0.1 nmol/L oxygen/min/1×106cells, simulated ischemia 3.1±0.1, adenosine 3.1±0.3, diazoxide 2.6±0.2,P<0.05). Cytoprotection, mitochondrial depolarization, and enhanced oxygen consumption were attenuated by the putative mitochondrial KATP-channel antagonist 5-hydroxydecanoate. The uncoupled phenotype in response to preconditioning was similarly observed in C2C12 myotubes. The present study suggests that modest mitochondrial uncoupling represents a unifying cellular response which may be important in directing preconditioning-mediated cytoprotection.