The O2 dependence of respiratory functions was studied in suspensions of isolated rat cardiac myocytes. Direct optical spectroscopy of the oxidation of cytochromes and oxygenation of Mb showed that cytochrome alpha 3 oxidation measured at 445-460 nm parallels Mb oxygenation; half-maximal values were at 8.0 and 8.5 microM, respectively. Thus there appears to be a close functional relationship between these components in the cells. The values are very high relative to comparable values for cytochrome alpha 3 oxidation in isolated rat heart mitochondria under state 3 conditions (0.43 microM) and isolated rat heart Mb (2.8 microM). Moreover, the measured values for half-maximal oxidation of cytochromes and oxygenation of Mb in the cells are sensitive to factors that alter the O2 consumption rate of the cells. These results indicate that mitochondrial respiration results in establishment of a gradient of O2 concentration from the suspending medium to the mitochondrial inner membrane. A portion of this gradient is between the suspending medium and the region occupied by Mb, and the remainder is between the region occupied by Mb and the inner mitochondrial membrane. The intracellular O2 gradient is an important factor in determining the O2 dependence of mitochondria in cells and hence may contribute to the O2 dependence of cardiac myocyte function in vivo.