To initiate studies on the transcriptional regulation of the human calcitonin receptor (hCTR) gene, a 4.9-kb hCTR promoter fragment was cloned and hCTR transcriptional initiation sites were mapped in human osteoclasts, kidney, and breast cancer cell line T47D. RT-PCR detected additional hCTR transcripts initiating at least 1 kb 5' to the transcripts mapped above, demonstrating that the hCTR gene is regulated by at least two separate promoters (hCTRP1 and hCTRP2). Transcripts initiating from the upstream promoter (hCTRP2) have a novel 5' untranslated region (5' UTR). Transfection of T47D breast cancer cells with hCTR promoter/luciferase deletion constructs demonstrated that the cloned 4.9-kb hCTR promoter fragment contains both hCTRP1 and hCTRP2 promoters. Fine deletion mapping of the more transcriptionally active hCTRP1 promoter demonstrated that only 97 bp of the hCTRP1 5' flanking region is required for the majority of its transcriptional activity.