Transcription of the calcitonin (CT) gene is down-regulated by vitamin D in normal and transformed thyroid C cells. DNA transfer techniques have been previously used to map and characterize a cAMP-induced enhancer at nucleotides -255 to -129 and an enhancer of basal transcription at -1060 to -905 in the CT 5' flanking DNA. The same methods were used to identify a negative response element for vitamin D. Deletion mutants of a genomic fragment of CT extending from nucleotides -1460 to +90 were attached to a promoterless GH gene and transfected individually into the medullary thyroid carcinoma cell line TT. CT nucleotides -1460 to -129 induced significant basal transcription of the GH reporter gene in TT cells. Basal transcription was elevated 3-fold to 4-fold by treatment with cAMP analog. The biologically active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3, had a minor (20%) inhibitory effect on basal transcription but inhibited more than 60% of the cAMP-induced transcription. We further investigated the cAMP-induced response and found that transcriptional activity of the downstream cAMP-induced enhancer was greatly synergized in the presence of the upstream enhancer of basal transcription. The latter enhancer contained three functional CANNTG sequences designated E1 (nucleotides -1060 to -1030), E2 (nucleotides -940 to -920), and E3 (nucleotides -920 to -900). E2 and E3 were essential for maximal cAMP-induced transcription. Detailed mapping of the vitamin D response showed that a minimum requirement for inhibition of the cAMP-induced enhancer by vitamin D was a sequence overlapping E3 (nucleotides -920 to -829). We conclude that a negative response element to vitamin D is located between nucleotides -920 and -829 in the CT 5' flanking DNA. It is possible that vitamin D inhibits transcription by interfering with the synergistic interaction between the cAMP-induced enhancer and the enhancer of basal transcription.