Chinese hamster V79-171 cells grown in vitro as asynchronous single cells, as plateau-phase cultures, and as multicell spheroids contianed subpopulations that differed in cell volume. These subpopulations could be isolated according to their sedimentation velocity at unit gravity. Asynchronous cells were found to sediment at a modal velocity of 17.7 mm/hr, whereas the modal sedimentation velocities of plateau and spheroid cells were found to be 16.0 and 15.1 mm/hr, respectively. When the composition of each subpopulation was determined using either [3H]thymindine incorporation or sensitivity to ionizing radiation as an index of cell cycle position, it was found that G1-like populations developed in both in vitro tumor models. Since the sedimentation velocity technique permits isolation of functionally different cell subpopulations after treatment with cytotoxic agents, it may now be possible to assay differential lethality when such agents are applied to cells growing in situ in a tumor-like situation.