A direct measurement of oxygen diffusion into mammalian cells in suspension has been made using a rapid-mixing technique and an oxygen-quenchable fluorescent probe. The emission from excited pyrene molecules located throughout the cytoplasm of Chinese hamster ovary cells, as determined by fluorescence photomicrography, is reduced in the presence of oxygen. This reduction in fluorescence intensity was monitored as a function of time following mixing of deoxygenated cells with oxygen-saturated phosphate-buffered saline (PBS) for times from 1 to 30 msec after mixing. When deoxygenated labeled cells were mixed rapidly with oxygen-saturated PBS, the half-time of quenching of the fluorescence was 3.7 msec. Analytical solutions were developed on the basis of diffusion into a spherical model cell and compared with experimental data. No effect of an unstirred sheath of water surrounding the cell could be detected, with a limit of sensitivity of about 1 /m. The data were well fitted on the basis of the diffusion model with a diffusion coefficient of 4 ? 2 x 10-6 cm2 sec-'. This value is fivefold lower than in water. The same value was inferred for diffusion in the local region of pyrene molecules through the equilibrium quenching constant determined from a Stern-Volmer relation. A comparison was made of these results with cell survival experiments when irradiations were performed within milliseconds of oxygen addition. Our data indicate that oxygen can diffuse to near-equilibrium values generally throughout a cell in only a few milliseconds.