The recently discovered epithelial calcium channels ECaC1 and ECaC2 are thought to play an important role in active calcium absorption in the intestine and kidney. Vitamin D-responsive elements (VDRE) were detected in the promoter sequence of human ECaC1 and regulation of ECaC by the steroid hormone 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) has been postulated. In this study we describe the structure of two murine ECaCs genes, each consisting of 15 exons localized on chromosome 6. Murine ECaC2 expression was found in many target tissues of 1,25-(OH)₂D₃, including skin and osteoblastic cells, while ECaC1 expression is confined to the kidney. By screening the murine promoter sequences, we detected a putative VDRE in ECaC1 and an estrogen response element in ECaC2. However, experiments in mice with a mutant, nonfunctioning vitamin D receptor showed that expression of ECaC1 in the kidney and of ECaC2 in duodenum is regulated by calcium levels, but not by 1,25-(OH)₂D₃. Also, estrogen-deficient ovariectomized (OVX) mice and OVX mice supplemented with estradiol showed unchanged duodenal ECaC2 expression compared with control mice. We conclude that ECaC expression in the kidney and the intestine is regulated by extracellular calcium but not by vitamin D or estrogen in vivo in mice.