A parathyroid hormone-related peptide (PTHRP) has been identified in human tumors associated with the syndrome of humoral hypercalcemia of malignancy. The PTHRP and parathyroid hormone (PTH) genes appear to have arisen by duplication and to represent members of a gene family. PTHRP mRNAs have been demonstrated in a number of normal tissues, but little is known concerning the regulation of PTHRP gene expression in any site. We studied PTHRP gene expression in TT cells, a human C-cell line which also produces calcitonin and calcitonin gene-related peptide. We found that both the synthetic glucocorticoid, dexamethasone, and the active vitamin D metabolite, 1,25-dihydroxyvitamin D3, decreased steady-state PTHRP mRNA levels in TT cells in a time- and dose-dependent fashion. The dexamethasone effect was completely blocked by the glucocorticoid antagonist RU-486. 24,25-dihydroxyvitamin D3 was found to be inactive. Neither dexamethasone nor 1,25-dihydroxyvitamin D3 appeared to influence PTHRP mRNA stability in TT cells, and both agents were shown by nuclear transcription run-off assay to decrease PTHRP gene transcription. These findings indicate that the PTHRP gene is under the transcriptional control of glucocorticoids and vitamin D in a cell line with prototypical neuroendocrine features.