Our previous work has shown that feeding 5 million international units (IU) of vitamin D3 to beef steers can produce tender strip loin and top round steaks. Our current experiment was designed to determine whether feeding two metabolites of vitamin D3, 25- hydroxyvitamin D3, and 1,25-dihysroxyvitamin D3, produces tender strip loin, top round, and top blade steaks more effectively than does supplemental vitamin D3 without leaving a substantial amount of residual vitamin D3 in muscle. Thirty-three continental crossbred steers were randomly allotted to one of four treatment groups. The first group was fed a placebo. The second group received 5 million IU of vitamin D3 each day for nine days and was slaughtered two days later. The third group received one dose of 125 mg of 25-hydroxyvitamin D3 four days before harvest, and the fourth group received one dose of 500 mg of 1,25- dihydroxyvitamin D3 three days before harvest. Blood samples were collected before treatment and at the time of slaughter for subsequent analysis of calcium, vitamin D3, 25-hydroxyvitamin D3, and 1,25-dihydroxyvitamin D3 concentrations in plasma. Steaks from the longissimus lumborum (strip loin) and semimembranous (top round) muscles were collected from each animal and aged for 8, 14, and 21 days, and steaks from the infraspinatus were collected and aged for 14 and 21 days. All steaks were analyzed for tenderness by Warner-Bratzler shear force determination. Concentrations of vitamin D3 in plasma were higher in vitamin D3- treated cattle (P < 0.0001). Concentrations of plasma 25-hydroxyvitamin D3 were increased in 25- hydroxyvitamin D3-treated cattle, but not as high as vitamin D3-treated cattle (P < 0.0001). 1,25- Dihydroxyvitamin D3 concentrations were higher in 1,25-dihydroxyvitamin D3-treated animals compared with all treatments (P < 0.0001). Supplementing steers with vitamin D3 increased the concentration of vitamin D3 and 25-hydroxyvitamin D3 in the meat of all muscles sampled (P < 0.0001). Supplementing steers with 25-hydroxyvitamin D3 increased the concentration of 25-hydroxyvitamin D3 in meat, but to an amount less than half that of cattle treated with vitamin D3. Warner-Bratzler shear force analysis showed that feeding 1,25- dihydroxyvitamin D3 did not significantly lower shear force values, but supplemental vitamin D3 and 25-hydroxyvitamin D3 produced longissimus lumborum and semimembranous steaks with lower shear force values (P < 0.06). Analysis of Western blots showed that longissimus lumborum and semimembranous steaks from cattle fed supplemental vitamin D3 and 25-hydroxyvitamin D3 (but not steaks from cattle fed 1,25- dihydroxyvitamin D3), had greater proteolysis of troponin T to a 30 kDa component.