Previous studies have demonstrated the involvement of a specialized, Na⁺-dependent carrier-mediated system for biotin uptake in mammalian intestine. The molecular identity of the carrier protein, the Na⁺-dependent multivitamin transporter (SMVT), has recently been identified. Upon characterization of transcript expression in the rat intestine, four distinct transcript variants (I-IV) due to heterogeneity at the 5'-untranslated region were found (Chatterjee NS, Kumar CK, Ortiz A, Rubin SA, and Said HM. Am J Physiol Cell Physiol 277: C605-C613, 1999). This finding raised the possibility that multiple promoters may be involved in driving the transcription of the SMVT gene. To test this possibility, we cloned the 5' regulatory region of the SMVT gene by genome walking. A 6.5-kb genomic DNA fragment was identified and sequenced. Three putative promoters (P1, P2, and P3) that were separated by exons of the four previously identified transcript variants were, indeed, found. P1 was found to contain multiple putative regulatory regions like GATA-1, AP-1, AP-2, and C/EBP, including several repeats of purine-rich regions and two TATA-like elements. P2 and P3 were GC rich and also revealed the presence of many putative regulatory elements including several SP-1 consensus sequences. The functional identity of each promoter and the minimal regions required for its function were established by the luciferase assay following transfection of rat-derived cultured intestinal epithelial IEC-6 cells. The highest functional activity of the cloned promoters was found to be in the order of P1 > P2 > P3. These findings represent the first characterization of the 5' regulatory region of any mammalian SMVT gene and should assist in the understanding of transcriptional regulation of this important gene.