Calbindin-D28k, a vitamin D-dependent calcium binding protein, plays a cardinal role in transport of calcium in kidney. Previous studies have demonstrated calbindin-D28k immunoreactivity in the distal nephron of mammalian kidney. However, it is well known that in most species including rat and human, there is a gradual transition from the distal convoluted tubule to the cortical collecting duct, and that the connecting segment do no tclearly demarcated, because of intermingling of distal convoluted tubule cells, connecting tubule cells, principal cells and at least two configurations of intercalated cells. In this study, to identify the cell types of calbindin-D28k-positive cells in distal nephron of rat kidney, we used double immunostaining with an antibody against calbindin-D28k and antibodies against thiazide sensitive Na+/Cl- cotransporter for distal convoluted tubule or H+-ATPase for intercalated cells. In the distal convoluted tubule, most of the distal convoluted tuble cells were calbindin-D28k-positive, whereas the intercalated cells were calbindin-D28k-negative. In the connecting tubule, 68% of the cells were calbindin-D28k-positive, and about 97% of the positive cells were connecting tubule cells and only 3% of them were intercalated cells. In the cortical collecting duct, and outer medullary collecting duct of outer stripe and inner stripe, only 8.6%, 11.8% and 4.4% of cells were weak positive for calbindin-D28k respectively. These weak positive cells in the collecting duct are mainly identified as intercalated cells. These findings indicate that calbindin-D28k is involved in not only transcellular transport of calcium but also processes regulating intracellular calcium in rat kidney.