Erythropoietin (EPO) is the key hormone for the differentiation and proliferation of erythroid progenitor cells. EPO is produced constitutively by the adult kidney to ensure normal erythropoiesis. Under conditions of reduced blood oxygen content EPO expression is substantially enhanced to increase erythropoiesis. Until today, cellular physiology of renal EPO production could not be studied due to the lack of an adequate human cell line. Here we present the human kidney cell line REPC (for renal Epo producing cells), established from an explanted human kidney which shows EPO gene expression and release of EPO protein in an oxygen-dependent manner. Hypoxic induction of EPO mRNA was first observed after 3 hours in 1% oxygen but then showed the typical transient increase with a peak in expression after 36 hours under continuous conditions of 1% oxygen. Bioactive EPO protein accumulated in the culture supernatant. Induction of EPO gene expression in REPCs critically depended on the activation of Hypoxia-inducible transcription factors (HIFs) with their oxygen-regulated HIF-alpha subunit and a constitutive HIF-beta subunit. SiRNA treatment revealed that expression of EPO was dependent on the activation of both transcription factor complexes HIF-1 and HIF-2. While HIF-1alpha accumulation was transiently induced with a maximal induction after 6 hours in hypoxia, HIF-2alpha was stably upregulated up to 24 h under hypoxic conditions. In addition, HNF4alpha was identified to be critically involved in hypoxia induced renal EPO expression. For the first time, with the human kidney cell line REPC we provide a powerful tool to study the cellular and molecular regulation of renal EPO production.