Retinoid X receptors (RXRs) heterodimerize with 1,25-dihydroxyvitamin D3 (VD) receptor (VDR), and play important roles in VD-regulated transactivation. VD acts on many tissues including kidney for the regulation of calcium homeostasis. In the kidney, the expression of VDR in the tubular cells has been well studied. In contrast, little is known about the localization and the functional significance of RXRs there. In order to elucidate these questions, we first performed immunohistochemical analyses of rat kidney using isoform-specific antimouse RXR antibodies we have previously reported. Interestingly, all RXR isoforms, predominantly RXR alpha, mainly localized to the proximal and the distal tubules, but not to the glomeruli. The serial section staining using anti-VDR antibody showed the colocalization of RXR alpha and VDR in those tubular cells. In order to elucidate the functional significance of endogenous receptors in the tubular cells, we next performed transient transfection studies using the tubular-cell derived Madin-Darby bovine kidney cells, which express both endogenous VDR and RXR. We transfected a reporter plasmid containing direct repeat 3 (DR3) sequence, to which only RXR/VDR heterodimer can bind, and found that VD and 9-cis retinoic acid, as well as VD and RXR selective agonist LG100153, had an additive effect for the DR3 transactivation. Taken together, we speculate that endogenous RXRs co-localize with VDR, and coregulate VD-dependent genes in the tubular cells of the kidney as RXR/VDR heterodimer.