Vertebrate histone gene promoters in many cases contain an upstream element, 5'dCCAAT, that has been implicated in modulating the efficiency of transcription of a broad spectrum of genes. We have previously isolated a nuclear factor (HiNF-B) that binds specifically to the CCAAT element of a cell cycle regulated human H1 histone gene. This factor shows similarities with other CCAAT box binding proteins in that it recognizes the same sequence but shows a distinct chromatographic behavior. In the present study, we have employed the gel retardation assay to demonstrate that HiNF-B is a cell cycle independent DNA binding protein that is conserved in both human and mouse cells. Using a series of reconstitution experiments with partially purified HiNF-B fractions, we show that this factor requires association of at least two components for site-specific binding. The composite structure of HiNF-B suggests that binding of at least some CCAAT elements in vertebrates may require cooperative interaction of CCAAT box binding proteins with other factors.