Thyroid hormones are important regulators of bone development and metabolism. We have demonstrated that tri-iodothyronine (T3) increased and 1,25-dihydroxyvitamin D3 (1,25D3) attenuated the T3-stimulated expression of osteocalcin (OCN) in the osteoblast-like cell line MC3T3-E1. By means of transfection of promoter-reporter gene constructs we investigated the basal and the regulated transcription of this gene by both hormones. We found that a 0.67 kbp and a 1.3 kbp fragment of the mouse OCN OG2 promoter containing two Runx2 binding sites were significantly more active than a smaller fragment containing only one Runx2 binding site. The longer promoter fragments showed a higher reporter gene expression when the transfected cells were treated with 10(-7) M T3. This expression was attenuated by 1,25D3 dose-dependently. These fragments contain a sequence homologue to the recently identified binding site for the 1,25D3 receptor (VDR) in the rat OCN promoter. Deletion of a part of the promoter containing this VDR response element-like sequence (VDRE) resulted in a higher basal expression but abrogated the regulation by T3 and 1,25D3. Electrophoretic mobility shift assays revealed that the deleted sequence was able to bind both in vitro-translated chicken thyroid hormone receptor (TR) and proteins from nuclear extracts that reacted with an antiserum against TR. From these data we conclude that the VDRE-like sequence of the OG2 promoter contains a thyroid hormone response element.