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Abstract Background: Both hypoxia and anemia stimulate erythropoiesis by stimulating Epo transcription and protein production. Developmental, tissue-specific, and environmental signals all contribute to the precise regulation of the Epo gene. Temporal and tissue-specific signals limit expression of the Epo gene primarily to cells in the fetal liver and adult kidney. The precise mechanisms regulating Epo gene expression during human fetal development are unclear. We sought to determine if regulation of the Epo gene occurs in some part through methylation. Using the demethylating agent 5-aza-2 deoxycytidine (DAC), we compared Epo mRNA expression in human fetal kidney and liver between 12 and 22 weeks gestation under normoxic and hypoxic conditions. Methods: Primary cell cultures from liver and kidney tissue ranging from 12-22 weeks were either treated with DAC or vehicle control for three days. After day three, each matched kidney and liver set were incubated at 1% (hypoxia) or 21% (room air) for eight hours in a 37 degree C incubator. RNA was harvested using TriZol, isolated, reverse transcribed, and quantification PCR was performed to measure Epo mRNA expression. Epo mRNA expression was normalized to an internal standard 18S rRNA in each duplex reaction. Results: Epo mRNA concentrations were much greater in liver than kidney at all gestations tested (p<0.001). A twenty-fold increase in Epo mRNA concentrations occurred when liver samples were exposed to hypoxia, however this increase was not enhanced by pretreating the samples with DAC. There was no statistical difference in Epo mRNA concentrations when kidney samples were exposed to hypoxia. There was a four-fold increase in Epo mRNA concentrations when kidney samples were pretreated with DAC, however this difference was not statistically significant. Conclusions: Demethylation of fetal kidney increased Epo mRNA expression, but not to the level of Epo mRNA expression measured in fetal liver. Methylation of fetal liver did not increase Epo mRNA expression under hypoxic conditions. We speculate that Epo gene expression in fetal kidney is regulated in part by methylation, and is developmentally regulated during mid-gestation.'