Cardiac failure appears rapidly during severe hypoxia and precedes a substantial reduction in adenosine triphosphate content. Reduced adenosine triphosphate turnover, in the presence of nearly normal content, may be the metabolic basis for contractile failure during hypoxia. To measure both the myocardial content and the turnover rates of high-energy phosphate compounds during hypoxia, we performed31P-nuclear magnetic resonance studies by placing a surface coil directly over the left ventricle in intubated rats that were instrumented for hemodynamic measurements and ventilated with either 21, 10, or 8% O2. Normoxia produced a hemodynamic and metabolic steady state for 4 hours and hypoxia for at least 60 minutes. Under normoxic ventilation (n= 10, mean ± SD), the arterial Poi was % ± 14, pH 7.38 ± 0.11, and systolic blood pressure 96 ± 8 mm Hg; under hypoxic ventilation with 10% O2(n =5), the arterial Po2was 57 ± 10, pH 7.39 ± 0.09, and systolic pressure 68 ± 10; and under hypoxic ventilation with 8% O2(n= 5), the Po2was 52 ± 7, pH 7.37 ± 0.04, and systolic pressure 51 ± 4. Hypoxic ventilation with 10 or 8% O2decreased the creatine phosphate content from 51.4 ± 5.4 μmol/g dry wt to 39.3 ± 5.4 and 45.6 ± 4.1 and depressed adenosine triphosphate slightly from 25.0 μmol/g dry wt to 21.8 ± 2.1 and 21.9 ± 1.0, respectively. High-energy phosphate turnover, measured as flux through the creatine kinase reaction, decreased from 22.7 ± 6.7 μmol/g dry wt/sec during normoxic ventilation to 13.7 ± 3.6 and 15.9 ± 2.6 during ventilation with 10 and 8% O2, respectively. Thus, the decreased turnover of high-energy phosphate compounds, not their tissue contents, may be the metabolic basis for contractile failure during hypoxia.