Osteocalcin is a major noncollagenous protein of bone regulated by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and is believed to be expressed only by differentiated osteoblasts. We introduced a 3.9-kilobase human osteocalcin gene promoter (hOCP)-chloramphenicol acetyltransferase (CAT) fusion gene into the germ line of mice. Examination of tissue extracts from these transgenic mice demonstrated that the expression of CAT was restricted to bone-associated tissues and the brain. Immunohistochemical staining of femur tissue sections using CAT antibodies localized the production of CAT protein to osteoblasts and maturing chondrocytes. Previous studies via transient transfection into osteoblast-like cells have identified a vitamin D response element approximately 500 basepairs up-stream of the hOCP capable of mediating 1,25-(OH)2D3 induction. As a consequence, regulation of the transgene was examined in homozygous transgenic lines for sensitivity to 1,25-(OH)2D3. Hormonal deficiency was created using a low calcium diet supplemented with 0.8% SrCl2 for 7 days and was restored in experimental mice by injection of 25 ng 1,25-(OH)2D3/day, ip, for 3 days. The low vitamin D3 diet decreased CAT activity several-fold in extracts from calvaria, femur, and brain compared to that in mice maintained on a normal diet, while 1,25-(OH)2D3 supplementation restored and enhanced CAT activity over control values. These data demonstrate that hOCP is sufficient to direct osteoblast-specific 1,25-(OH)2D3-sensitive gene expression in mice in addition to the unexpected regulatable expression in brain tissue.