The mammalian β-like globin gene family has served as an important model system for analysis of tissue-and developmental stage-specific gene regulation. Although the activities of a number of regulatory proteins have been implicated in the erythroid cell-specific transcription of globin genes, the mechanisms that restrict their expression to discrete stages of development are less well understood. We have previously identified a novel regulatory element (PRE II) upstream from the human embryonic β-like globin gene (ε) that synergizes with other sequences to confer tissue- and stage- specific expression on a minimal ε-globin gene promoter in cultured embryonic erythroid cells. Binding of an erythroid nuclear protein (PRE II- binding factor [PREIIBF]) to the PRE II control element is required for promoter activation. Here we report on some of the biochemical properties of PREIIBF, including the characterization of its specificity and affinity for DNA. The embryonic and adult forms of PREIIBF recognize their cognate sequences with identical specificities, supporting our earlier conclusion that they are very similar proteins. PREIIBF binds DNA as a single polypeptide with an M(r) of ~80,000 to 85,000 and introduces a bend into the target DNA molecule. These results suggest a mechanism by which PREIIBF may contribute to the regulation of the embryonic β-like globin gene within the context of a complex locus.