The vitamin D-dependent calcium-binding protein (CaBP) was localized by immunocytochemistry in the rat and human kidney. In both species 80% of the cells lining the distal convoluted tubules contained CaBP. In the connecting segment and the initial collecting tubule of rat kidney, 50% of the cells was positive; in the outer medullary collecting duct only 15% was positive. In the human kidney, collecting ducts in medullary rays contained 50% positive cells, whereas in the rest of the medulla no positive cells were found. The CaBP-positive cells were identified as principal or clear cells by immunoelectron-microscopy, using the protein A-gold technique. Mitochondria-rich dark cells were negative. In principal cells, CaBP immunoreactive sites were found throughout the cytosol and the nuclear euchromatin. No preferential labeling of cellular membranes was found. The data show that CaBP-positive cells are present in tubular regions that are important in regulating the final excretion of calcium. However, the subcellular distribution of CaBP does not suggest a role in the initial transmembrane transport of Ca2+ but rather indicates an involvement in processes regulating intracellular calcium.