The functional interaction between the human epsilon globin promoter and an erythroid-specific transcription enhancer, 5' HS-2, has been analyzed by transient expression assay. While stepwise deletion of DNA sequences between -852 and -122 had only small effects, removal of the CACC box at position -111 greatly decreased epsilon-globin promoter activity, as well as its response to the enhancer function of 5' HS-2 in erythroid cells. Our data demonstrated that the three ubiquitous promoter elements, the CACC, CCAAT, and TATA boxes, of the epsilon-globin-encoding gene together form a minimal promoter that would interact efficiently with 5' HS-2, and that at least the CACC box is an essential functional component of this enhancer-promoter interaction.