Although distal regulatory regions are frequent throughout the genome, the molecular mechanisms by which they act in a promoter-specific manner remain to be elucidated. The human beta-globin locus constitutes an extremely well-established multigenic model to investigate this issue. In erythroid cells, the beta-globin locus control region (LCR) exerts distal regulatory function by influencing local chromatin organization and inducing high-level expression of individual beta-like globin genes. Moreover, in transgenic mice expressing the entire human beta-globin locus, deletion of LCR-hypersensitive site 2 (HS2) can alter beta-like globin gene expression. Here, we show that abnormal expression of human beta-like globin genes in the absence of HS2 is associated with decreased efficacy of pre-initiation complex formation at the human epsilon- and gamma-promoters, but not at the beta-promoter. This promoter-specific phenomenon is associated with reduced long-range interactions between the HS2-deleted LCR and human gamma-promoters. We also find that HS2 is dispensable for high-level human beta-gene transcription, whereas deletion of this hypersensitive site can alter locus chromatin organization; therefore the functions exerted by HS2 in transcriptional enhancement and locus chromatin organization are distinct. Overall, our data delineate one mechanism whereby a distal regulatory region provides promoter-specific transcriptional enhancement.