The gene encoding the human calbindin-D9k has been cloned and the complete sequence established. The gene spans about 5.5 kilobases and is localized on the X-chromosome, consists of three exons and carries four Alu repeats. The promoter and 1300 base-pairs of 5' flanking region have been characterized. Besides a TATA box and two CAAT-like motifs a sequence related to a vitamin D response element was detected about 1.1 kilobases upstream from the promoter. A sequence positioned 50 nucleotides downstream from the promoter showed extensive homology to the estrogen response element at the same location within the rat calbindin-D9k gene. Two essential nucleotides within this region are changed when the rat and human sequences are compared. The human element failed to bind the estrogen receptor as determined by gel retardation assay. It is proposed that a two-nucleotide change within this region causes the gene to lack expression in human uterus and possibly placenta.