The concentration of oxygen within cells is important in many physiological and pathological processes, but such oxygen-dependent processes are generally studied as a function of the concentration of extracellular oxygen, due to a lack of suitable methods. Using a newly developed technique based on ESR spectroscopy, we show that respiration stimulation of a cell suspension can result in a significant difference between average intracellular and extracellular concentrations of oxygen. These results indicate that studies of oxygen-dependent phenomena in cells may require measurement of intracellular oxygen concentrations and imply that there are mechanisms in cells that restrict the free diffusion of oxygen.