The bone-specific osteocalcin (OC) gene is transcribed only after completion of proliferation in normal diploid calvarial-derived osteoblasts during extracellular matrix mineralization. In contrast, the OC gene is expressed constitutively in both proliferating and nonproliferating ROS 17/2.8 osteosarcoma cells. To address molecular mechanisms associated with these tumor-related modifications in transcriptional control, we examined sequence-specific interactions of transactivation factors at key basal and hormone-responsive elements in the OC gene promoter. In ROS 17/2.8 cells compared to normal diploid osteoblasts, the absence of a stringent requirement for cessation of proliferation to support both induction of OC transcription and steroid hormone-mediated transcriptional modulation is reflected by modifications in transcription factor binding at (i) the two primary basal regulatory elements, the OC box (which contains a CCAAT motif as a central core) and the TATA/glucocorticoid-responsive element domain, and (ii) the vitamin D-responsive element. Particularly striking are two forms of the vitamin D receptor complex that are present in proliferating osteoblasts and osteosarcoma cells. Both forms of the complex are sensitive to vitamin D receptor antibody and retinoic X receptor antibody. After the down-regulation of proliferation, only the lower molecular weight complex is found in normal diploid osteoblasts. Both forms of the complex are present in nonproliferating ROS 17/2.8 cells with increased representation of the complex exhibiting reduced electrophoretic mobility that is phosphorylation-dependent.