The human zeta-globin promoter contains a strong positive regulatory element in the 5' flanking region, designated the zeta-globin upstream regulatory element (URE). In this study, we define the minimal sequences required for URE function and characterize the associated protein-DNA interactions. Deletion experiments show that the URE spans a 60 bp region located between 220 and 279 bp 5' to the transcription start site. Further subdivision of this region shows that multiple cis acting sequences are present. Electrophoretic mobility shift assays demonstrate that the erythroid transcription factor GATA-1 binds a site at -230, and Sp1 and an unidentified factor bind a CCACC site at -240. The unidentified CCACC factor is distinct from two other CCACC factors, EKLF and BKLF/TEF-2. A third complex contains a novel DNA-binding activity that interacts with a site in the -269 to -255 region, designated URE binding factor (URE-BF). This factor is present in K562 cells that express zeta-globin, but is absent in the OCIM1 cell line, a human erythroid cell line that does not express zeta-globin. URE-BF appears to interact with a GATA factor, since formation of the URE-BF complex can be prevented by the presence of unlabeled oligonucleotides containing GATA sites. Finally, increasing the distance from the -230 GATA site to the two upstream sites causes a progressive decrease in zeta-globin promoter activity. There is no indication of a requirement for GATA-1 to be on the same side of the DNA helix as the other upstream factors. These results show that zeta-globin promoter function is highly dependent on a 60 bp region to which at least three different factors bind. Two of these factors may represent DNA-binding proteins not previously identified as important for regulation of globin gene expression. It is likely that these factors interact physically to create a functional regulatory unit.