Calbindin-D9k is expressed in the cytoplasm of intestinal cells, where it is critical for dietary calcium absorption. Two striking aspects of the expression of this gene are its vitamin-D dependency and regional differences in expression, with high levels only in duodenum. We report studies of the human calbindin-D9k promoter. Differences between the reported sequences of the human calbindin-D9k promoter were first clarified before undertaking a functional analysis of this sequence. Studies of the rat gene have indicated that several transcription factors, including the caudal-related homeobox factor (CDX-2), hepatic nuclear factor-4 and CCAAT-enhancer-binding protein alpha (C/EBPalpha), could interact with elements in the promoter. Although these elements are conserved in the human gene, we show here that their intestinal distribution makes them unlikely to be critical positive factors. The calbindin-D9k gene contains multiple potential binding sites for homeobox transcription factors; one of these, known as IPF-1 or PDX-1, co-localizes in the intestine with calbindin-D9k. We show in gel-shift assays that the sequence within a putative vitamin-D-response element in the human calbindin-D9k promoter can bind expressed IPF-1/PDX-1 protein, although we cannot confirm binding of the vitamin-D-receptor protein. CDX-2 binds to the region around the TATA box, as in the rat gene, and may act as a negative factor in the distal intestine. Transfection studies in Caco-2 and MCF-7 cells with heterologous reporter vectors containing up to 1303 bp of the gene showed that this functioned as a weak promoter and indicated the presence of suppressor sequences, but did not show vitamin-D responsiveness. This indicates that other elements are also needed for the control of human calbindin-D9k expression.