Isokinetic-gradient sedimentation employing a shallow linear gradient of Ficoll in tissue culture medium was used to isolate erythroid progenitor cells (CFU-e) from mouse bone marrow. Following gradient sedimentation, 34% of the total nucleated cells and 48% of the CFU-e applied to the gradient were recovered, and three distinct modal populations of CFU-e could be distinguished. The slowest-migrating population did not require exposure to exogenous erythropoietin in order to form erythroid colonies in vitro. The other two modal populations of CFU-e required exposure to exogenous erythropoietin for differentiation. One of these, constituting 64% of the hormone-dependent CFU-e recovered, migrated with the bulk of the marrow cells, whereas the other migrated ahead of the bulk of the marrow cells. This latter population, which contained 34% of the CFU-e, was recovered with 11% of the marrow cells, representing a twofold to threefold enrichment. BFU-e migrated more slowly than the erythropoietin-dependent CFU-e. Resedimentation studies suggested that the two erythropoietin-dependent CFU-e populations were distinct modal populations. When cells from the fastest-migrating population of erythropoietin-dependent CFU-e were cocultured with unseparated marrow cells, a further twofold to threefold enhancement of erythroid colony formation was obtained. Comparison of isokinetic-gradient sedimentation with discontinuous and continuous albumin density-gradient sedimentation revealed that isokinetic-gradient sedimentation was a more efficient method than the former and a more rapid method than the latter for isolating CFU-e from mouse bone marrow.