Tubular reabsorption of Mg⁺⁺ is mediated by the tight junction protein, paracellin-1, which is encoded by the gene PCLN-1 (CLDN16) and exclusively expressed in the kidney. Tubular Mg⁺⁺ reclamation is modulated by many hormones and factors. The aim of this study was to define regulatory elements essential for renal tubular cell-specific expression of human PCLN-1 (hPCLN-1) and to explore the effect of Mg⁺⁺ transport modulators on the paracellin-1 gene promoter. Endogenous paracellin-1 mRNA and protein were detected in renal cell lines OK, HEK293, and MDCT, but not in fibroblast cell line, NIH3T3. A 7.5kb hPCLN-1 5'-flanking DNA sequence along with seven 5'-deletion products were cloned into luciferase reporter vectors and transiently transfected into the renal and non-renal cells. The highest levels of luciferase activity resulted from transfection of a 5'-flanking 2.5kb fragment (pJ2M). This activity was maximal in OK cells, was orientation-dependent, and was absent in NIH3T3. Mg⁺⁺ deprivation significantly increased pJ2M-driven activity in transfected OK cells, whereas Mg⁺⁺ load decreased it when compared with conditions of normal Mg⁺⁺. Deletion analysis along with electrophoretic-mobility-shift-assay demonstrated that OK cells contain nuclear proteins, which bind a 70bp region between -1633 and -1703 of major functional significance. Deleting this 70bp segment, which contains a single peroxisome-proliferator-response-element (PPRE), or mutating the PPRE, caused a 60% reduction in luciferase activity. Stimulating the 70bp sequence with 1,25(OH)₂VitD decreased luciferase activity by 52%. This effect of 1,25(OH)₂VitD was abolished in the absence of PPRE or in the presence of mutated PPRE. We conclude that the PPRE within this 70bp DNA region may play a key role in the cell-specific and regulatory activity of the hPCLN-1 promoter. Ambient Mg⁺⁺ concentration and 1,25(OH)₂VitD may modulate paracellular, Expression and Regulation of the Human Paracellin-1 Gene 3 paracellin-1-mediated, Mg⁺⁺ transport at the transcriptional level. 1,25(OH)₂VitD exerts its activity on the hPCLN-1 promoter likely via the PPRE site.