Critical intracellular myocardial oxygen tension was determined by optical spectroscopic measurement of myoglobin oxygen saturation in crystalloid-perfused guinea pig hearts. Accurate end-point determinations of the maximally oxygenated and deoxygenated myoglobin were made. Hearts were subjected to a steady decrease in perfusate oxygen tension while left ventricular developed pressure, maximal left ventricular dP/dt, myocardial oxygen consumption, lactate release, and adenosine release were measured as indices of myocardial function. Intracellular myoglobin was found to be only 72% saturated under baseline conditions with an arterial oxygen tension of >600 mmHg at 37°C. Baseline intracellular oxygen tension was 6.3 mmHg. Myocardial oxygen consumption was decreased by 10% when the oxygen tension fell to 5.7 mmHg, and cardiac contraction decreased 10% when oxygen tension was 4.1 mmHg. Adenosine release and, finally, lactate release began to increase at sequentially lower oxygen tensions. The present results indicate that the buffer-perfused guinea pig heart at 37°C has an intracellular oxygen tension just above the threshold for impaired function.