AbstractThe concentration of oxygen within cells is important in many physiological and pathological processes, but such oxygendependent phenomena are generally studied as a function of the concentration of extracellular oxygen, due to a lack of suitable methods. Electron parmamagnetic resonance (EPR) oximetric techniques offer an attractive alternative to make such measurements. Previous EPR oximetric studies of extracellular‐intracellular oxygen gradients have been hindered, however, by the fact that separate samples, prepared in slightly different ways, were required for individual measurements of extracellular and intracellular oxygen concentrations. In this study we demonstrate a technique that allows simultaneous measurement of intracellular and extracellular oxygen concentrations in a single sample: extracellular measurement is achieved using positively charged nitroxides encapsulated in liposomes, while intracellular oxygen is determined using a membrane‐permeable nitroxide along with an extracellular broadening agent. Application of this system to the measurement of oxygen concentrations in suspensions of rat myoblast cells gave results which are consistent with nonsimultaneous measurements and which show substantial extracellular‐intracellular oxygen gradients in these rapidly respiring