Fetal hemoglobin production in adult human erythroid progenitors can be influenced by exogenous factors. In this report, we examine the effects of erythropoietin (Epo) and hydroxyurea (HU) in serum-free media on fetal (gamma-) globin mRNA levels when present during the first week of peripheral blood burst-forming unit-erythroid (BFU-E) culture. Fetal calf serum (FCS) is known to elevate gamma-globin mRNA levels in BFU-E culture and has been shown to do so even when added after the first week of culture. Removal of FCS from the first week of our BFU-E cultures did not significantly reduce gamma-globin mRNA levels, in agreement with these earlier findings. Addition of Epo to BFU-E cultures 4 days before addition of FCS led to a significant drop in gamma-globin mRNA levels; Epo addition also led to a significant increase in the number of erythroid bursts. Culture conditions, including Epo in serum-free liquid medium for 1 week followed by plating in semisolid medium containing FCS and Epo, resulted in low gamma-globin mRNA levels, similar to levels found in vivo, and high burst numbers. These conditions were used to demonstrate significant elevation of gamma-globin mRNA levels by HU present during the first week of BFU-E culture. Our findings suggest that gamma-globin mRNA levels can be influenced early in erythropoiesis by Epo and HU.