Calcitonin (CT), whose secretion from thyroid glands is regulated by increases in the concentration of extracellular Ca(2+), is a well-known hormone that regulates calcium homeostasis. However, the molecular mechanisms underlying the gene expression dependent on Ca(2+) have not been clarified. The downstream regulatory element (DRE) antagonist modulator (DREAM) was recently identified as a Ca(2+)-dependent transcriptional repressor. In the present study, we investigated the possible involvement of DREAM in the regulation of CT gene expression and secretion. A luciferase assay using TT cells, a thyroid carcinoma cell line, showed that a particular region in the CT gene promoter repressed the promoter activity under basal conditions but induced the activity when the Ca(2+) concentration was increased. We found two DRE sequences in a region located upstream from the transcription start site. Gel retardation assay confirmed that DREAM bound to the CT-DRE and also indicated that DREAM bound to the DRE in a Ca(2+)-dependent manner. We generated stable transfectants of TT cells with wild-type or mutant DREAM, which lacked the responsiveness to Ca(2+) changes. In contrast to the wild type, overexpression of the mutant DREAM inhibited the increase in CT secretion induced by a calcium ionophore. The addition of forskolin to increase cAMP activated the CT promoter, probably by the interaction of DREAM with cAMP-responsive element binding proteins, independent on the activation by Ca(2+). Together, these results suggest that DREAM plays an important role in human CT gene expression in a Ca(2+)- and cAMP-dependent manner.