In osteoblast-like cells in culture, the human osteocalcin gene promoter basal activity is repressed by glucocorticoids, reflecting the repression of serum osteocalcin concentrations noted in syndromes of glucocorticoid excess. 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], the active hormonal form of vitamin D, induces the osteocalcin promoter through a vitamin D response element (VDRE), and glucocorticoids also repress the vitamin D-induced promoter. This study addresses the role of a glucocorticoid receptor (GR) binding site overlapping the TATA box of the osteocalcin promoter, which had been proposed as a negative glucocorticoid response element (nGRE), invoking a steric interference mechanism of glucocorticoid repression. Confirmation of the role of the nGRE in regulating basal activity was obtained using promoter constructs containing a TATA box swap. However, a minor component of repression of 1,25-(OH)2D3 induced activity remained in the absence of the nGRE. In addition, glucocorticoid repression of the human osteocalcin promotor was shown to be cell line specific. This result is not compatible with a simple model of repression and suggests the existence of unidentified cell-specific factors that are involved in the repression event. Repression of the osteocalcin promoter was compatible with a composite model involving both the nGRE site and glucocorticoid regulation of factors that bind the vitamin D response element.