In an attempt to stimulate Hb F synthesis in baboons by means other than erythropoietic stress, we considered the possibility that an agent that inhibits methylation of CpG sequences in DNA may be effective. 5-Azacytidine, a cytosine analogue that cannot be methylated, is such an agent. Animals whose packed red cell volume was maintained at approximately 20% by bleeding were given 10 daily intravenous injections of the drug (6 mg/kg) in 12 days. Hb F levels in these animals started to increase on day 5 of this regimen and peak levels, which were 6-30 times higher than those produced by bleeding alone, occurred 5-7 days after the last dose of the drug. In animals previously identified as genetically "high" or "low" Hb F responders, the maximal Hb F levels were 70-85% and 35-40% respectively. In dose-response studies 5-azacytidine given daily at 3-4 mg/kg produced maximal Hb F increases. The drug did not correlate the percentage (number) of Hb F-containing cells (F cells) beyond the maximal number achieved by bleeding alone and thus its main effect was to increase Hb F per F cell. The finding that Hb F synthesis can be modulated to such a high degree by a drug may have therapeutic implications--e.g., in sickle cell anemia, in which stimulation of Hb F synthesis may prevent sickling.