Cells expressing the bone-specific osteocalcin (OC) gene exhibit two DNase I hypersensitive sites within the proximal (nt -170 to -70) and distal (nt -600 to -400) promoter. These sites overlap elements that independently or in combination contribute to basal and vitamin D-stimulated OC gene transcription. Here we address mechanisms that participate in control of chromatin remodelling at these sites. By applying nuclease digestion and indirect end-labeling or by combining intranuclear footprinting and ligation-mediated PCR, we investigated the effects of nuclear protein hyperacetylation on both chromatin organization and transcriptional activation of the OC gene in bone-derived cells. We report that chromatin hyperacetylation blocks vitamin D stimulation of OC transcription and prevents a key transition in the chromatin structure of the OC gene which is required for formation of the distal DNase I hypersensitive site. This transition involves interaction of sequence-specific nuclear factors and may be required for the ligand-dependent binding of the vitamin D receptor complex, which results in transcriptional enhancement.