Colony assays are now available to study erythroid differentiation at three different levels. Mixed hemopoietic colonies represent progeny of pluripotent progenitors (CFU-GEMM). Erythroid bursts and colonies are derived from early (BFU-E) and late precursors (CFU-E) that are committed towards erythropoiesis. The three different types of colonies were examined for their content of fetal hemoglobin (HbF) by radioimmune assay and/or immunofluorescence. A comparison of both methods showed the radioimmune assay to be more sensitive. Frequency analysis revealed more than 90% of all mixed hemopoietic colonies positive for HbF. The proportion was significantly lower for erythroid bursts and even further reduced for erythroid colonies. Quantitative assessment established a range of HbF concentrations for erythroid colonies between 0 and 500 pg. For erythroid bursts, concentrations between 0 and 5000 pg were measured. The amount of HbF did not correlate with the size of the examined bursts. The addition of stimulators other than erythropoietin, provided in our culture system by media conditioned with leukocytes in the presence of phytohemagglutinin (PHA-LCM), increased the frequency of HbF-containing bursts. However, the quantitative distribution of HbF was not affected. These results are consistent with the view that more primitive erythroid progenitors are more likely to give rise to HbF-containing progeny. The similarity in distributions of HbF concentrations suggests a regulatory mechanism responsible for HbF synthesis that is intrinsic to the progenitor cells and appears to be independent of cell division.