In a phosphate-vitamin D-deficiency rachitic model in rats, potassium pyroantimonate was employed as a histochemical stain for calcium at the ultrastructural level in the costochondral growth plates. In the control plates, there was a shift of calcium from an intracellular, mainly mitochondrial location in the top part of the zone of hypertrophic cells to an extracellular, mainly matrix-vesicle location in the bottom part of the zone of hypertrophic cells. In the rachitic plates, mitochondria and cell membranes throughout the bottom of the hypertrophic zone remained heavily loaded with calcium. After treatment with phosphate was started, the mitochondria and cell membranes at the bottom of the hypertrophic zone rapidly lost calcium and matrix calcification began. Thus, in the normal growth plate, matrix calcification begins at the level in the plate where mitochondria lose calcium; in rickets, the matrix does not calcify and mitochondria do not lose calcium; and in healing rickets, calcification begins in the matrix at the bottom of the hypertrophic zone as the mitochondria at that level lose calcium. These findings support the hypothesis that mitochondria play an important role in matrix calcification.