Nuclease hypersensitive sites were mapped in the proximal promoter of the osteocalcin gene, which is expressed only in bone cells exhibiting the mature osteoblast phenotype. Nuclei from proliferating and confluent rat osteosarcoma (ROS) 17/2.8 cells were subjected to DNase I digestion, and hypersensitivity was assayed by the indirect end-labeling method, using osteocalcin gene probes. Hypersensitive sites were detected in two promoter domains: -590 to -390, which spans the vitamin D responsive element, and -170 to -70, which spans the TATA box and the CCAAT-containing OC box domain. Together, these elements regulate basal and vitamin D enhanced osteocalcin gene transcription. We observed a parallel relationship between the intensity of bands representing the hypersensitive sites and the extent to which the osteocalcin gene is transcribed. Both in confluent cultures and in response to vitamin D, when osteocalcin transcription was upregulated, the hypersensitive bands were significantly intensified. Additionally, the bands were decreased under conditions that downregulate osteocalcin gene transcription. A functional relationship between the presence of hypersensitive sites and osteocalcin gene transcription is further supported by the absence of hypersensitivity in nonosseous cells that do not express osteocalcin, although these proliferating cells exhibited hypersensitivity in a cell cycle regulated histone gene promoter. Our results suggest the involvement of chromatin structure in transcriptional responsiveness of the osteocalcin gene to physiologic modulation.