The promoter of the calbindin-D 9k (CaBP9k) gene, previously analyzed in transgenic mice, contains all of the information necessary for expression of a transgene similar to the endogenous gene and also for an appropriate response to vitamin D. In the present study we first investigated the role of a putative vitamin D-responsive element (9k/VDRE), located at nucleotides− 489 to −445 on the rat CaBP9k promoter gene, using transgenic mice. As expected, the pattern of transgene expression in mice carrying this putative VDRE mutated in its whole promoter context was similar to that in mice bearing the wild-type sequence. These transgenic mice also responded to 1,25-dihydroxyvitamin D₃ in the same way as those bearing the wild-type transgene and as those carrying a transgene with a large deletion (from −2894 to −117) eliminating the putative 9k/VDRE. Thus, the putative 9k/VDRE is not required for the control of rat CaBP9k gene expression by vitamin D in vivo. We also found that responsiveness to 1,25-dihydroxyvitamin D₃ depends on the site at which the transgene is integrated into the host genome, in a tissue-specific manner. These data together with the fact that vitamin D-responsive sequences are present in a two-module region (from −3731 to −2894 and/or −117 to +365) and that this region does not contain any classical VDRE show that the CaBP9k gene is submitted to a nonconventional control by vitamin D.