This electron microscope study describes the subcellular occurrence and distribution of immunoreactive calbindin-D9K in the trabecular metaphyseal and compact cortical bone of normal rats, rachitic vitamin-D-deficient rats, and rachitic rats given 1,25-(OH)2D3. Undecalcified bones were embedded in Lowicryl K4M and calbindin-D9K antigenicity was detected by the protein A-gold method. Immunoreactive calbindin-D9K was localized in the cytoplasm and cell processes of osteoblasts and osteocytes. Immunoreactive calbindin-D9K was also found within matrix vesicles and calcifying matrix vesicles, where it lay over the needle-shaped crystallites, at the apparent site of initial crystal formation, but not along the whole crystallites. In fully mineralized bone it occurred at the same site, over the crystallites. Calibindin-D9K was vitamin-D-dependent in the osteoblasts and matrix vesicles, where its presence was correlated with the reappearance of crystallites in 1,25-(OH)2D3-treated vitamin-D-deficient rats. This suggests that immunoreactive calbindin-D9K is involved in mineral deposition in bone matrix vesicles. Abnormal intracellular calcification associated with calbindin-D9K antigenicity in the osteoblasts of 1,25-(OH)2D3-treated vitamin-D-deficient rats indicates that immunoreactive calbindin-D9K may also play a part in abnormal intracellular mineral deposition.