Thirty-two patients with aplastic anemia (AA) have been studied using the long-term bone marrow culture (LTBMC) system. Of these patients, 26 had been treated with immunosuppressive therapy including antilymphocyte globulin (ALG) with or without androgens or high-dose methyl prednisolone. The remaining six patients either required no treatment or were studied before therapy was begun. Thirty-one of 32 patients (96%) had defective hematopoiesis in LTBMC with little or no evidence for the generation of primitive progenitor cells. The only exception was a patient with spontaneous recovery of aplasia in whom the defect was less marked. Crossover LTBMC experiments were performed in 23 cases by inoculating (1) patient marrow hematopoietic cells that had been depleted of adherent cells onto preformed, irradiated, normal stromas to assess the proliferative capacity of the hematopoietic cells, and (2) normal marrow hematopoietic cells that were depleted of adherent cells onto preformed, irradiated stromas from patients with AA to assess stromal function. Results of these experiments demonstrated a hematopoietic defect in all patients that was independent of the degree of hematologic recovery after ALG therapy. Only one patient had a probable stromal defect and this coexisted with a defect in the regenerative capacity of hematopoietic cells. We conclude that LTBMC is a sensitive method for detecting and defining the hematopoietic failure in AA. We suggest that the defective hematopoiesis present in all patients studied may be important in the pathogenesis of clonal evolution in AA.