The persistence of fetal hemoglobin in many patients with deletion type β thalassemias and the expression patterns of human globin genes in transgenic mice suggest that γ-to β-globin gene switching results primarily from competition of γ- and β-globin genes for interaction with the β-globin locus control region (LCR). To define regulatory sequences that are essential for the competitive advantage of the γ gene at early developmental stages, stable transgenic mouse lines were produced with LCR γ-β constructs containing deletions of γ5'-flanking DNA. All constructs contained the full 22 kb LCR, a 4.1 kb β-globin gene and a γ-globin gene with 1348, 383, 202, 130, 72 or 52 bp of 5'-flanking sequence. Primer extension analysis of yolk sac, fetal liver and blood RNA from these lines demonstrated that a region between-202 and-130 of the human γ-globin gene promoter was required to suppress β-globin gene expression at early developmental stages. Four transcription factor binding sites within this region [GATA(p), Oct1, GATA(d) and CACCC] were mutated independently in LCR γ-β constructs and transgenic mouse lines were produced. Only the γ CACCC box mutation resulted in high levels of β-globin gene expression in early embryos. These results demonstrate that the γ CACCC box of the human γ-globin gene plays a critical role in human β-globin gene developmental specificity. The data also suggest that γ CACCC box binding factors mediate LCR-γ interactions which normally enhance γ-globin and suppress β-globin gene expression in fetal erythroid cells.