Two human neuroblastoma (NB) cell lines, SH-SY5Y and Kelly, were found to express the gene for erythropoietin (EPO) in an oxygen (O₂)-dependent manner. However, NB cells had maximal production of EPO with lower partial pressure of O₂ values than the well-characterized hepatoma cell line HepG2. This maximal EPO expression was preceded by accumulation of the O₂-sensitive α subunit of the heterodimeric transcription-factor complex hypoxia-inducible factor 1 (HIF-1). Western blot analysis revealed that the amount of the β subunit of HIF-1, identical to aryl hydrocarbon receptor nuclear translocator 1 (ARNT1), and the homolog ARNT2 increased in nuclear extracts from SH-SY5Y cells exposed to anoxia. In neuronal cells, ARNT1 and ARNT2 can form a heterodimer with HIF-1α, generating a functional HIF-1 complex. Using the hypoxia response element of the human EPO enhancer, we conducted electrophoretic mobility shift assays that showed accumulation and binding of HIF-1 complexes containing both ARNT1 and ARNT2 in NB cells. In addition to the HIF-1 complex, hepatocyte nuclear factor 4α (HNF4α) was found to be indispensable for hypoxia-induced EPO gene expression in hepatoma cells. Western blot analysis and polymerase chain reaction assessment showed that NB cells express neither HNF4α nor the splicing variant HNF4α7 and thus express EPO in an HNF4α-independent manner. Together, SH-SY5Y and Kelly cells may provide a new in vitro model for studying the mechanism of tissue-specific, hypoxia-inducible EPO gene expression.