We examined the effects of acute and/or chronic hypokalemia on responses to 30 min of hypoxia and recovery in the isolated, perfused heart model. We found that both acute hypokalemia and chronic hypokalemia impaired contractility [expressed as maximum slope of pressure increase over time (dP/dt): 501 ± 49 and 529 ± 48 vs. 1,302 ± 118 mmHg/s, P < 0.01] and recovery of ATP concentrations (determined with ³¹P NMR spectroscopy: 30 ± 6 and 40 ± 10 vs. 67 ± 5% initial, P < 0.05) at 30 min of recovery. Moreover, the combination of acute hypokalemia and chronic hypokalemia had additive effects (dP/dt 166 ± 15 mmHg/s and ATP 21 ± 7% initial, both P < 0.01). We also measured cytosolic calcium with surface fluorescence spectroscopy after indo 1 loading. Acute hypokalemia and acute hypokalemia + chronic hypokalemia increased cytosolic calcium (averaged throughout the cardiac cycle) during and after hypoxia (390- to 460-nm ratio at 30 min of recovery: 0.46 ± 0.07 and 0.65 ± 0.07 vs. 0.18 ± 0.03, P < 0.01), whereas control and chronic hypokalemia hearts had only small changes with hypoxia and recovery. Finally, when we examined mitochondria isolated from hearts perfused under experimental conditions, we found that chronic hypokalemia-alone mitochondria and chronic hypokalemia + acute hypokalemia mitochondria had marked impairment of state 3 respiration compared with control hearts (52 ± 13 and 50 ± 9 vs. 128 ± 10 natm · min¹ · mg protein¹ with succinate as substrate, P < 0.01), whereas acute hypokalemia mitochondria demonstrated only subtle changes. These data suggest that both acute hypokalemia and chronic hypokalemia impair cardiac responses to hypoxia. The mechanism may involve impairment of calcium metabolism, but cytosolic calcium alterations do not explain all of the metabolic and functional effects of acute hypokalemia and chronic hypokalemia in the setting of hypoxia.