Erythropoietin (Epo), the primary regulator of the production of erythroid cells, acts by binding to a cell surface receptor (EpoR) on erythroid progenitors. We used deletion analysis and transfection assays to examine the transcription control elements in the 5' flanking region of the human EpoR gene. In erythroid cells most of the transcription activity was contained in a 150 bp promoter fragment with binding sites for transcription factors, AP2, Sp1 and the erythroid specific GATA-1, but lacking a TATA sequence. Mutation analyses indicated that both Sp1 and GATA-1 were required for high level EpoR expression. The EpoR promoter could be transactivated by GATA-1 in K562 erythroid cells and non- erythroid HeLa cells when activity was low. In OCIM1 erythroid cells with high constitutive levels of EpoR, the EpoR promoter activity was high and GATA-1 was able to transactivate only the Sp1 mutated EpoR promoter with low activity. These data suggest that while GATA-l may be responsible for tissue specific expression of hEpoR, transcription activity depends on coordination between Sp1 and GATA-1 with other cellular factors, including possibly other Sp1-like binding proteins, to provide high-level, tissue- specific expression. We had previously demonstrated that a human EpoR genomic fragment containing about 2 kb of 5' flanking region was able to direct EpoR expression in hematopoietic tissue and in brain. The EpoR promoter and 5' flanking region were linked to the beta-galactosidase gene and assayed in transgenic mice. The 1778 bp 5' flanking region was able to express the reporter gene transiently in the embryonic brain as was observed for the endogenous EpoR gene, while a 150 bp promoter fragment provided reporter gene expression in the adult brain. Neither construct activated reporter gene expression in hematopoietic tissue but did provide expression in testis which is high in GATA-1 but endogenous EpoR expression is silent. These studies indicate that while the 5' flanking region is able to provide some regulation of brain expression, additional 3' sequences are required for activation of EpoR in hematopoietic tissue.