The effect of prolonged settling times (up to 2 h), in high- and low-phase columns, on the cell partition ratios measured and on the separability of cell populations was examined. With closely related cell populations, modelled by rat erythrocytes in which subpopulations of red blood cells of distinct age were labeled isotopically, it was found that partitioning proceeds over the entire time period examined as evidenced by the continuous change in relative specific activity of cells in the top phase as the partition ratio falls. In control cell sedimentation experiments in top phase there was almost no change in the quantity of cells present when vertical settling (i.e., high-phase columns) was used and no separation of specific subpopulations was found. In the horizontal settling mode the initially higher cell partition ratio, as compared to vertical settling, decreased to a greater extent with longer time intervals; a given purity of cells only being obtained at a lower partition ratio than in the vertical settling mode. Cell sedimentation in top phase was appreciable with time in the horizontal settling mode but did not result in a separation of cell subpopulations. The effect of relative cell partition ratios and sizes in high- and low-phase columns on the efficiency of separation was examined by use of rat or sheep 51Cr-labeled red cells mixed with an excess of human unlabeled erythrocytes. Rat and sheep red cells are appreciably smaller than human erythrocytes. Rat red cells have higher, and sheep red cells lower partition ratios than human erythrocytes. With vertical settling, over a 2-h period, there is no appreciable contribution to the change in relative specific activities by cell sedimentation. However, the more rapid sedimentation of the larger human red cells has, with time, a measurable effect on the relative specific activities obtained during cell partitioning when run in the horizontal mode: enhancing the rat-human and diminishing the sheep-human cell separations. Partitioning cells in high-phase columns is of advantage with respect to increasing separation efficiency and virtually eliminating the influence of other physical parameters (e.g., cell size). Since the cell partitioning process continues for long periods of time, yielding ever-lower partition ratios with increasing proportions of cells with higher P values, a time may be selected which balances desired relative cell purity and yield.