To analyse the 5'-flanking sequences required for the tissue specific transcription of the human erythropoietin receptor (hEpo-R) gene, a DNA region spanning nucleotides -1050 to +135 relative to the transcription initiation site (+1) was explored. Our studies indicate that a minimum promoter (-76/+33) containing GATA and SP1 binding sites at positions -45 and -20 is not sufficient to confer erythroid specific expression to a reporter gene. Erythroid specificity of the promoter was observed either with the (-1050/+33 construct) which contains a cluster of Alu repetitive elements or with the addition of the 135 bp down to the transcription initiation site (-76/+135 construct) which exert a negative control on the promoter activity with a major effect in non erythroid tissues. The latter region can be subdivided on two distinct domains: the +1/+78 region that exerts a positive effect and the +79/+135 region that has a negative effect on the Epo-R promoter activity measured by CAT assays. The first region contains three CANNTG motifs, whereas the second contains an SP1/CACCC motif at position +85. These findings reveal a complex regulation of the hEpo-R gene and provide a working model useful to explain how the minimal promoter, containing GATA/SP1, can be positively and negatively regulated during erythroid differentiation.