The DNA flanking the 5′ sequence of the mouse 1α-hydroxylase gene has been cloned and sequenced. A TATA box has been located at −30 bp and aCCAAT box has been located at −79 bp. The gene’s promoter activity has been demonstrated by using a luciferase reporter gene construct transfected into a modified pig kidney cell line, AOK-B50. Parathyroid hormone stimulates this promoter-directed synthesis of luciferase by 17-fold, whereas forskolin stimulates it by 3-fold. The action of parathyroid hormone is concentration-dependent. 1,25-Dihydroxyvitamin D₃ does not suppress basal promoter activity and marginally suppresses parathyroid hormone-driven luciferase reporter activity. The promoter has three potential cAMP-responsive element sites, and two perfect and one imperfect AP-1 sites, while no DR-3 was detected. These results indicate that parathyroid hormone stimulates 25-hydroxyvitamin D₃-1α-hydroxylase by acting on the promoter of the 1α-hydroxylase gene.