We have analyzed the reactivation of fetal hemoglobin (HbF) synthesis under rigorous in vitro conditions, ie, in mature erythroblasts generated by erythroid burst-forming units (BFU-E) stringently purified from normal adult peripheral blood and grown in fetal calf serum(FCS)- free semisolid or liquid phase culture. In clonogenetic dishes, graded amounts of c-kit ligand (KL) were added together with saturating levels of erythropoietin (Ep) and variable amounts of interleukin-3 and granulocyte-macrophage colony stimulating factor (IL-3/GM-CSF), ie, high or low level, or no IL-3/GM-CSF addition. In all conditions, KL induced a sharp, dose-dependent increase in the percentage of F cells and HbF content from nearly normal levels (< 10% and < 2.5%, respectively, at 0.1 and 1 ng/mL) up to 40% to 50% and 10% to 15% at 100 to 200 ng/mL. This increase was not associated with significant differences of burst number or stage of maturation at the time of analysis (as evaluated on the basis of percent mature erythroblasts and Hb content per cell). However, the KL-induced reactivation of HbF synthesis was strictly and directly correlated with a sharp increase of colony size, ie, cell number per burst. Addition of large amounts of IL- 3 and GM-CSF (10 to 100 U and 1 to 10 ng/mL, respectively) significantly potentiated the KL-induced reactivation of HbF, as compared with low levels (0.1 U and 0.01 to 0.1 ng) or no addition of these growth factors: this increase was highly significant at low KL doses (ie, 1 to 10 ng/mL). Single-burst analysis showed that the KL- induced HbF reactivation occurs homogeneously in the erythroid colonies within each of these culture conditions. We have analyzed the effect of KL in liquid phase BFU-E culture treated with the IL-3/GM-CSF/Ep combination at sequential times until terminal erythroid maturation: KL causes a sharp increase in the percentage of F cells and HbF content in all stages of maturation, whereas the IL-3/GM-CSF/Ep combination alone has a markedly lower effect. These results suggest that KL plays a key role in the reactivation of HbF synthesis in adult life, whereas IL- 3/GM-CSF potentiate this effect at low KL levels. The KL-induced HbF reactivation is seemingly related to an enhanced proliferation of erythroid progenitors in the erythropoietic differentiation pathway.