The proximal promoter of the human H4 histone gene FO108 contains two regions of in vivo protein-DNA interaction, Sites I and II. Electrophoretic mobility shift assays using a radiolabeled DNA probe revealed that several proteins present in HeLa cell nuclear extracts bound specifically to Site I (nt-125 to nt-86). The most prominent complex, designated HiNF-C, and a complex of greater mobility, HiNF-C', were specifically compatable by an Sp1 consensus oligonucleotide. Fractionation of HiNF-C using wheat germ agglutinin affinity chromatography suggested that, like Sp1, HiNF-C contains N-acetylglucosamine moieties. Two minor complexes of even greater mobility, designated HiNF-E and F, were compatable by ATF consensus oligonucleotides. A DNA probe carrying a site-specific mutation in the distal portion of Site I failed to bind HiNF-E, indicating that this protein associated specifically to this region. UV cross-linking analysis showed that several proteins of different molecular weights interact specifically with Site I. These data indicate that Site I possesses a bipartite structure and that multiple proteins present in HeLa cell nuclear extracts interact specifically with Site I sequences.