To understand the role of CCAAT-binding factor (CBF) in transcription in the context of chromatin-assembled DNA, we used regularly spaced nucleosomal DNA using topoisomerase IIalpha (topo IIalpha) and alpha2(1) collagen promoter templates, which were subsequently reconstituted in an in vitro transcription reaction. Binding of CBF to the nucleosomal wild-type topo IIalpha promoter containing four CBF-binding sites disrupted the regular nucleosomal structure not only in the promoter region containing the CBF-binding sites but also in the downstream region over the transcription start site. In contrast, no nucleosome disruption was observed in a mutant topo IIalpha promoter containing mutations in all CBF-binding sites. Interestingly, CBF also activated transcription from nucleosomal wild-type topo IIalpha promoter. In this experiment, a promoter containing one wild-type CBF-binding site was activated very weakly, whereas the promoter containing mutations in all sites was not activated by CBF. A truncated CBF that lacked the glutamine-rich domains did not activate transcription from nucleosomal wild-type topo IIalpha promoter but disrupted the nucleosomal structure about as much as did the binding of full-length CBF. Two nucleosomal mouse alpha2(1) collagen promoter DNAs, one containing a single and the other containing four CBF- binding sites, were also reconstituted in an in vitro transcription reaction. None of the nucleosomal collagen promoters was activated by CBF. However, both of these collagen promoters were activated by CBF when the transcription reaction was performed using naked DNA templates. Binding of CBF to the nucleosomal collagen promoter containing four binding sites disrupted the nucleosomal structure, similarly as observed in the topo IIalpha promoter. Altogether this study indicates that CBF-mediated nucleosomal disruption occurred independently of transcription activation. It also suggests that specific promoter structure may play a role in the CBF-mediated transcription activation of nucleosomal topo IIalpha promoter template.