We have analyzed the synthesis of fetal hemoglobin (HbF) in the differentiated erythroid colonies produced by erythropoietin-responsive committed erythroid progenitors in plasma cultures of human marrow and peripheral blood. Two independent biochemical techniques. carboxymethyl-cellulose column chromatography of 3H-leucine-Iabeled globin. and polyacrylamide gel electrophoresis of 59Fe-labeled hemoglobin were used to quantitate the hemoglobin(s) synthesized in replicate cultures containing varying concentrations of the erythropoiesis-stimulating hormone erythropoietin. Excellent quantitative agreement between these two methods was observed. Sensitivity of these assay methods was such that 5% fetal hemoglobin could be detected. In separate studies of three normal adult males, we observed little or no detectable fetal hemoglobin synthesis in the differentiated colonies derived from marrow erythroid progenitors over a range of erythropoietin concentration that produced increasing numbers and size of erythroid colonies. Peripheral blood erythroid progenitors of these same individuals were studied in cultures containing 0.5 U/mI and 2.0 U/mI of erythropoietin. At both these erythropoietin doses, fetal hemoglobin was synthesized at concentrations varying between 9% and 39% of the adult hemoglobin. Cytoplasmic RNA was isolated from the differentiated progeny of peripheral blood erythroid progenitors to permit analyses using radioactive cDNA probes specific for human a-, /9-, and ‘yglobin mRNA. Results from the RNAexcess cDNA liquid hybridization experiments also indicated that ‘y-globin mRNA was present in peripheral blood BFU-Ederived erythroid colonies at a level comparable to the amount of ‘y-globin chain synthesis observed. In contrast. erythroid colonies derived from marrow and blood erythroid progenitors of three hematologically abnormal individuals synthesized approximately equal amounts of fetal hemoglobin in plasma culture. Once again, the level of HbF synthesis was unrelated to erythropoietin concentration in culture. We conclude that the erythroid progenitor cells ordinarily found in the peripheral blood (F progenitors) are programmed to produce differentiated erythroid colonies that synthesize fetal hemoglobin. The great majority of normal bone marrow erythroid progenitors are not programmed to produce colonies that synthesize hemoglobin F. but under conditions of anemic stress, the population of F progenitors within the marrow expands sufficiently to populate erythroid cultures with colonies that produce considerable quantities of hemoglobin F.