The effect of supplementing diets with various levels of vitamin D3 to provide 0, 0.5, 1, and 5 million IU/(steer x d) for 8 d before slaughter on the mineral content and localization of Ca in LM and muscle fragments was studied during the postmortem aging process. Twelve feedlot steers of three biological types were given access to the four levels of vitamin D for 8 d before slaughter. Differential centrifugation techniques were used to determine the concentrations of minerals relative to protein in different muscle fragments on d 3 and 21 postmortem. Electron microscopy visualization of bound Ca indicated that vitamin D3 mobilized Ca from the sarcoplasmic reticulum and transverse tubule system into the myofibrils. Bound Ca was concentrated near the Z-line at the A-band/I-band juncture within the sarcomere. Supplementing steers with 1 and 5 million IU/(steer x d) of vitamin D3 increased (P < 0.05) Ca, P, and Mg concentrations per unit of protein in the cytosol. Soluble cytosolic Ca concentrations were greater (P < 0.05) on d 21 than on d 3 postmortem only when steers were supplemented with 5 million IU/d. Concentrations of Ca, P, and Mg in isolated tissues were increased (P < 0.05) in nuclei and myofibrilar proteins by supplementing steers with 1 and 5 million IU/ (steer x d) of vitamin D3. All supplemental vitamin D3 treatments also increased (P < 0.001) Mg concentrations in the cytosol, regardless of aging treatment, and increased Mg concentrations (P < 0.04) within the mitochondria at d 3 postmortem. Thus, supplementation of feedlot steers with vitamin D3 at levels of 0.5 to 5 million IU/(steer x d) increased Ca concentrations within respiring muscle, resulting in increased bound tissue Ca concentrations. When the respiring muscle was converted to meat, the increased bound tissue Ca resulting from vitamin D3 treatment released Ca concentrations into the cytosol during aging (P < 0.05). Results of this study indicate that vitamin D3 supplementation increased total cytosolic Ca, P, and Mg concentrations in meat.