To characterize the persistent abnormalities of hematopoiesis in aplastic anemia (AA) after immunosuppression with antilymphocyte globulin (ALG), we analyzed the quantity, phenotype, and growth properties of hematopoietic progenitor cells in 13 patients who received ALG treatment. Flow cytometry (FACS) revealed a deficiency of CD34+ cells in bone marrow (BM) of all patients. This deficiency was most severe (40-fold) in 4 patients in AA relapse. In 9 patients in remission, CD34+ cells were reduced 2-10-fold and showed no correlation with the ALG-induced improvement of peripheral blood cell counts. The proportion of CD34+ cells carrying c-kit receptors was abnormally low (2-10-fold below normal) in 5 of 13 AA patients. These patients also displayed low levels of c-kit mRNA by reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, the CD34+ cell population was almost completely depleted of CD34+CD38- early hematopoietic progenitors in all AA patients. The proportion of CD34+ cells expressing lineage differentiation antigens CD33, CD71, and CD45RA in AA was increased, as compared to control BM. Formation of hematopoietic colonies by FACS-purified CD34+ cells was nearly absent in 4 relapsed patients, normal in 4 of 9, and decreased (up to 10-fold) in 5 of 9 patients in remission. The degree of impairment of colony-forming ability by AA progenitors correlated well with the reduction of CD34+ c-kit+ cells. The best proliferative response of CD34+ cells was observed in the presence of stem cell factor and, in some cases, fit3 ligand. Our results indicate that the disease process in AA depletes immature BM progenitors, thus providing a plausible explanation for persistent defects in colony-forming ability and long-term regenerative capacity of AA marrow after immunosuppression. Analysis of the immunophenotypes and the proliferative properties of purified progenitors may be useful for estimating degree of hematopoietic recovery in ALG-treated patients.