Erythropoietin (Epo) functions as a stimulating factor, resulting in proliferation of erythrocyte precursors and triggering differentiation to a mature erythrocyte or red blood cell. Therefore, it is likely that the cellular sensitivity to Epo greatly changes during the course of erythroid cell differentiation in parallel with the erythropoietin receptor (Epo-R) gene expression. We have recently cloned in the genomic DNA of human Epo- R; in its 5' flanking region, there are potential regulatory sequences specific to the erythroid cell lineage. Therefore we speculate that 5' flanking region of the Epo-R gene is important for its regulation of expression at the transcriptional level. In order to examine the roles of these motifs on transcriptional regulation, we made plasmids containing deletions of the 5' flanking region with a reporter gene (luciferase) and transfected these constructs into cultured cells, OCIM-1 (high expressed Epo-R) and HeLa (no expressed Epo-R). Analysis of expression in OCIM-1 cells by luciferase assay indicates that - 150 bp from the initiation start site is very critical for transcription. This region has an inverted GATA motif and short stretches of sequences which share homology with other transcriptional factors such as SP1. However, we did not detect any conspicuous events in other regions such as strong enhancers or silencers in the distal 5 region extending to about 2 kb 5' of the cap site. On the other hand, the result of experiments in HeLa cells demonstrated that the longest fragment (-937 to -1778) has transcriptional activity. These results demonstrate that non Epo-R expressing cell lines contain positive trans-acting factors that can activate the Epo-R enhancer and promoter in transient assays, yet probably the endogenous Epo-R in this cell is repressed. We suggest that repression of the chromosomal Epo-R gene in HeLa cells is a result of mechanisms that restrict accessibility of enhancer and promoter elements to trans- regulators possibly via changes in chromatin structure. In the erythroid system, Epo-R is likely regulated at multiple levels including chromatin structure, transcription regulation, and polypeptide translocation to the cell membrane resulting in modulation of Epo-R expression during erythroid maturation.