To determine the effect of supplemental dietary vitamin D3 on concentration of vitamin D and its metabolites in milk, 20 Holstein cows were assigned to four groups and fed either 0, 10,000, 50,000, or 250,000 IU of vitamin D3/d beginning approximately 2 wk prepartum and continuing through wk 12 of lactation. Samples of blood plasma and milk were assayed for concentrations of vitamin D, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D. Only the daily dosage of 250,000 IU caused significant increases of concentrations of vitamin D or 25-hydroxyvitamin D in plasma. Concentrations of vitamin D and 25-hydroxyvitamin D in milk were approximately equal and averaged .2 ng/ml. Little 1,25-dihydroxyvitamin D and no 24,25-dihydroxyvitamin D could be detected in milk from any of the four treatment groups. Cows fed 250,000 IU of vitamin D3/d produced milk containing 54 IU of vitamin D activity per liter, whereas unsupplemented cows produced milk containing 17 IU/L. Oral supplementation with up to 250,000 IU of vitamin D3/d does not increase effectively vitamin D activity of milk.