We have shown that the minimal enhancer fragment present in the 3'-flanking region of the human beta-globin gene contains four regions that bind nuclear proteins in vitro. By using gel mobility shift and DNase I footprinting assays, we were able to show that each of these regions binds an erythroid-cell-specific nuclear factor which we name NF-E1. This factor is present in erythroid cells at different developmental stages of globin gene expression. The recognition sequence of this protein (A/C Py T/A ATC A/T Py) is also present in the intragenic enhancer and the promoter of the beta-globin gene as well as in the promoter of other erythroid-cell specific genes. In addition to NF-E1, each of the four binding regions interacts with at least one other protein factor.