A cAMP-induced enhancer was previously mapped to nucleotides -255 to -85 of the calcitonin (CT) gene 5'-flanking DNA. To determine the functional cis-acting elements within this region, we transfected medullary thyroid carcinoma (MTC) cells with CT 5'-flanking DNA/GH fusion genes containing potential cAMP-responsive elements and assessed their transcriptional activities with and without cAMP. In CT-expressing MTC cells (the TT line), we identified by deletions and point mutations three transcriptionally active motifs: a cAMP-responsive element (CRE), TGACGTCA, at -253 to -246, and a hybrid site containing a CRE-like element (CREL; TGACCTCA, -169 to -162) adjacent to an equally transcriptionally active octamer (O) sequence (ATG-CAAAT, -161 to -154). These three motifs acted synergistically and their transcriptional activity was completely dependent on cAMP. In HeLa cells their synergistic activity was more constitutive than cAMP induced, whereas in CT-negative MTC cells (the RO-D81-1 line) these motifs were inactive. Gel mobility shift assays with antibodies against CRE-binding protein (CREB) and activating transcription factor 1 (ATF-1) showed that both CREB and ATF-1 interacted with the CRE in MTC cells, whereas in HeLa cells only ATF-1 bound to the CRE. Specific binding to the CREL/O motif was detectable in extracts from tumors induced by injection of TT cells but not in extracts from any of the three cultured cell lines. We conclude that cAMP-induced transcription of the CT gene is modulated in a cell-specific manner by the CRE and the CREL/O elements.