We have previously shown that the active form of vitamin D, 1,25(OH)₂D₃, has both genomic and rapid nongenomic effects in heart cells, however the subcellular localization of the vitamin D receptor (VDR) in heart has not been studied. Here we show that in adult rat cardiac myocytes the VDR is primarily localized to the t-tubule. Using immunofluorescence and Western analysis we show that the VDR is closely associated with known t-tubule proteins. Radioligand binding assays using ³H-labeled 1,25(OH)₂D₃ demonstrate that a t-tubule membrane fraction isolated from homogenized rat ventricles contains a 1,25(OH)₂D₃ – binding activity similar to the classic VDR. For the first time we show that cardiac myocytes isolated from VDR knockout (KO) mice show accelerated rates of contraction and relaxation as compared to wild type (WT), and that 1,25(OH)₂D₃ directly effects contractility in the WT but not the KO cardiac myocyte. Moreover, we observed that acute (5 min) exposure to 1,25(OH)₂D₃ altered the rate of relaxation. A receptor localized to t-tubules in the heart is ideally positioned to exert an immediate effect on signal transduction mediators and ion channels. This novel discovery is fundamentally important in understanding 1,25(OH)₂D₃ signal transduction in heart cells and provides further evidence that the VDR plays a role in heart structure and function.