Upstream of the chicken beta-globin gene cluster are four DNase I-hypersensitive sites (HS1-4). Hypersensitive sites located upstream of the mammalian beta-globin clusters have enhancer activity and mediate position-independent gene expression. In contrast, a region inside the chicken cluster has enhancer activity and mediates position-independent expression. Here we investigate the function of the chicken upstream sites, which are different from the mammalian ones in sequence, number, and distance from the genes. Each was tested for its effect on reporter gene expression in transfected primary erythroid cells. HS2 and HS3 (4.4 and 6.4 kilobases upstream of rho-globin) showed significant enhancer activity while HS1 and HS4 (1.6 and 11 kilobases upstream of rho-globin) did not. A 237-base pair region of HS2 contained the sequences necessary for enhancer activity. Proteins from erythroid extracts bound HS2 in seven different regions; six of these sites were characterized. GATA-1 bound to four of the sites. Each site contributed to the enhancer activity of HS2. Two other sequences bound proteins that may be related to Sp1 and erythroid krüppel-like factor. Surprisingly, mutations in these elements, which disrupted protein binding, did not affect enhancer activity. Thus, the observed enhancer activity of HS2 is due to the four GATA sites. The existence of multiple GATA sites in both chicken HS2 and the mammalian upstream sites may be due to evolution from a common element with preservation of only very short sequences or to convergent evolution. These observations highlight the crucial role for GATA proteins in globin regulation.