Marrow from 28 nonleukemic individuals was separated by adherence to glass or plastic into nonadherent (NA) and adherent populations. The NA populations were found to be more dependent for colony formation in culture on added colonystimulating activity (CSA) than unseparated marrow suspensions, and therefore proved useful for CSA assays. Quantitative reconstitution procedures were used to assay CSA-producing cells. Either increasing numbers of irradiated unseparated marrow, or adherent cells derived from varying numbers of marrow cells, were used to restore colony-forming efficiency to NA populations. Assay procedures for CSA-producing cells were applied to four patients with acute leukemia prior to treatment. In all four instances, a defect in CSA-producing cells was demonstrated.