Although severe deficiency of bioactive vitamin D (1,25OH2D) causes rickets, mild insufficiency of the hormone, known as hypovitaminosis D, is responsible for the occurrence of secondary hyperparathyroidism and osteoporosis. To clarify the pathophysiology of the disease, we studied the negative feedback effect of 1,25OH2D and its precursor 25OHD on the transcriptional activity of parathyroid hormone (PTH) gene using the PT-r parathyroid cell line. We found that PT-r cells express endogenous 1alpha-hydroxylase as well as PTH mRNAs. We also found the potent suppressive effect of physiological concentration of 25OHD on the transcriptional activity of PTH gene. A similar effect was obtained with 1,25OH2D but only with pharmacological concentration. Interestingly, the effect of 25OHD was completely abolished when the cells were treated with 1alpha-hydroxylase inhibitor ketoconazole. These results suggest that the negative feedback regulation of vitamin D on PTH gene transcription occurs not by the end-product 1,25OH2D but by its prohormone 25OHD via intracellular activation by 1alpha-hydroxylase within the parathyroid cells.