Magnetic interactions between dissolved oxygen and nitroxide radical spin probes lead to broadening of the ESR lines. We have used a closed-chamber method based on this property to determine the maximum rate of O2 uptake per cell (Vmax per cell) in cultured mammalian cells. A suitable spin probe and a cell suspension are mixed in an aerated medium, and the rate of disappearance of dissolved O2 is measured. The effects of temperature, pH, and microwave power on the determination of dissolved oxygen in solution were studied. For asynchronous Chinese hamster ovary cells, oxygen uptake is 3.8 X 10(7) oxygen molecules per cell per sec and appears to be enzymatically limited at oxygen concentrations greater than 10 microM. About 5-10 X 10(5) cells were used for each measurement, making it possible to study mitotically synchronized cells. Using this method, we have found that Vmax per unit cell volume changes during the cell cycle of Chinese hamster ovary cells from a minimum in mitosis to maxima in both G1 and late S phases. Advantages and limitations of spin probes for studying the O2 uptake of intact cells are discussed.