This review summarizes current knowledge on the distribution of the vitamin-D-dependent, calcium-binding protein, calbindin-D9k, an indicator of 1,25(OH)2D3 action, in mineralized tissues, with emphasis on the cellular and subcellular distribution of the protein. Light and electron microscopic immunolocalization studies have shown that calbindin-D9k is present in cartilage and bone. In cartilage, it is restricted to the mature chondrocytes. In bone, it is present in osteoblasts and osteocytes. In both, its synthesis is vitamin D dependent. While calbindin-D9k is a strictly cytosolic protein in soft tissues, it has a specific distribution in mineralized tissues and may even be extracellular. Hence, it is restricted to the lateral edges of the longitudinal septa in the epiphyseal cartilage, the same area in which matrix vesicles are found. Calbindin-D9k lies within the matrix vesicles and is also found in the matrix vesicles near the mineralizing front of trabecular and compact bone. It seems to remain in position over the crystallites formed from the matrix vesicles in fully calcified cartilage and in heavily mineralized bone. The protein is also present in teeth, in the ameloblasts of incisors and molars but not in odontoblasts. The data suggest that calbindin-D9k is required for mineral nucleation in the matrix vesicles of epiphyseal cartilage and bone. The synthesis and distribution of calbindin-D9k in normal and rachitic normal bone and cartilage indicate that vitamin D has a direct action on mineralizing tissues.