The effect of the cholesterol content of the plasma membrane on the intracellular concentration of oxygen in Chinese hamster ovary (CHO) cells and their mutants was investigated by EPR oximetry. Total and free cholesterol content was significantly higher in 25 RA CHO cells as compared to wild-type and M 19 CHO cells, with most of the free cholesterol in normal and mutant CHO cells located in the plasma membrane. The plasma membrane cholesterol content also was altered by various biochemical means, and the effect on the oxygen gradient was studied. Comparing the three cell lines, the gradient was larger with increased content of cholesterol in the plasma cell membrane. This result also is supported by an additional increase in the oxygen gradients with the incorporation of additional cholesterol in the plasma membrane and a decrease in the oxygen gradient when the cholesterol was depleted from the plasma membrane. The results indicate that the concentration of cholesterol in the plasma membrane can be an important factor for the magnitude of the oxygen gradient observed across the cell membrane.