U sing microscopic immunodiffusion assays and microdensitometric analysis of pericellular immunoprecipitate, the percentage of nucleated erythrocytes containing fetal hemoglobin (FNRBC) and the mean picograms of fetal or adult hemoglobin per nucleated erythrocyte (picograms HbF/NRBC, picograms HbA/NRBC) were assayed in 14-d-old colonies (bursts) derived from peripheral blood erythroid progenitors. In the peripheral blood of 11 normal adults only 2.2±0.5% (mean±SE) erythrocytes contained HbF whereas pooled bursts from the same subjects revealed a 13-fold increase in the percentage of FNRBC (29.6±3.9%). In culture both the picograms HbF/NRBC (5.2±0.4) and the picograms HbA/NRBC (27.7±1.5) are increased ∼20% above the mean in vivo levels in NRBC from normal bone marrow aspirates. Analysis of each of 58 bursts from one subject demonstrated that FNRBC are present in all bursts and range from 5.0 to 95.0% of the total NRBC per burst. The percent FNRBC in each burst was neither correlated with picograms HbF/NRBC per burst nor with picograms HbA/NRBC per burst. Individual subcolonies from one burst in each of two subjects demonstrated between 3 and 81% FNRBC.These findings indicate that first, the increase in HbF production in culture is primarily due to increased production of the number of cells containing HbF, not to increased picograms HbF/NRBC. Second, all 14-d bursts contain some FNRBC. Third, just as seen in vivo, the picograms HbF/cell and the number of cells that contain HbF are independently regulated in culture. Fourth, commitment to produce HbF in vitro continues after subcolony formation in 14-d-old bursts. Augmentation of HbF production in culture therefore closely resembles that seen in acute erythroid stimulation in vivo.