High level expression of many eukaryotic genes is achieved through the action of distal regulatory sequences or enhancers. We have utilized the interaction between the erythroid-specific enhancer in hypersensitivity site 2 (HS2) of the human β-globin locus control region and the globin gene promoters as a model to elucidate the mechanisms governing promoter/enhancer interactions. HS2 contains a 400-base pair core element consisting of tandem AP1/NF-E2 motifs flanked by binding sites for multiple ubiquitous and erythroid-specific factors. We have compared the enhancer activity of this core element with a synthetic enhancer lacking the factor binding sites flanking the AP1/NF-E2 motif (HS2(M)). In fetal/erythroid K562 cells, enhancement of a linked γ-promoter was significantly greater with wild-type HS2 than with HS2(M). In contrast, the increase in β-promoter activity in these cells was equivalent with either enhancer fragment. Truncation of the binding site for the fetal/erythroid-specific stage selector protein in the γ-promoter abolished the additional enhancer activity of HS2. Similarly, insertion of the stage selector protein site into the β-promoter boosted enhancer activity observed with HS2 but not HS2(M). In adult erythroid MEL cells, enhancement of a linked β-promoter was significantly greater with HS2 than with HS2(M). This effect was dependent on the binding of the adult stage-specific factor, erythroid Kruppel-like factor, to the β-promoter. Taken together, this data suggests that the stage-specific factors binding the proximal globin promoters and the factors flanking the AP1/NF-E2 motif of HS2 act in synergy.