Large-scale separation of mixtures of mammalian cells was obtained with the A-1X zonal centrifuge rotor and density gradients consisting of Ficoll dissolved in modified Eagle's MEM suspension-culture medium. The cells remained viable as tested by plating efficiency or by motility observed with time-lapse photography. Rabbit thymocyte and HeLa cell mixtures were separated with 99 and 89 per cent purity, respectively. Mixtures of thymocytes and suspension-cultured, human acute leukemia cells (Roswell Park strain LKID) were separated with 93 and 91% purity, respectively. HeLa cells were isolated 92% pure from a mixture with horse leukocytes. A book of charts giving the sedimentation position and velocity versus time of cells in the A rotor under standard conditions of gradient composition, angular velocity, and temperature was prepared with the use of a computer program based on the differential sedimentation equation. The charts are used to estimate the centrifugation time necessary for maximum separation of cells. The success achieved in separating mixtures of cells points to the future possibility of large-scale fractionation of solid tissues, especially tumor tissues, into preparations cf viable cells of a single type.