Human milk has been found to contain 40 to 50 IU/l of vitamin D activity. This was determined by measuring stimulation of intestinal calcium transport in the rat, an assay not subject to the errors inherent in the rat line test or calcification assay. Five vitamin D metabolites were then isolated using a combination of conventional chromatography on Sephadex LH-20 and Lipidex 5000 followed by high-performance liquid chromatography. 24,25-Dihydroxyvitamin D and 1,25-dihydroxyvitamin D were measured using binding protein assays and were found to be present at very low levels. These dihydroxylated metabolites do not contribute significantly to the total vitamin D activity. Vitamins D2 and D3 were found to be present at concentrations of 338 and 41 ng/l, respectively. This is equivalent to 14 to 16 IU/l of vitamin D activity. Human milk contains 163 ng/l of 25-hydroxyvitamin D3, which gives about 33 IU/l of vitamin D activity. Thus 25-hydroxyvitamin D3 accounts for about 75% of the biological activity observed in the calcium transport assay. Vitamin D2, vitamin D3, and 25-hydroxyvitamin D3 are responsible for more than 90% of the total vitamin D activity present. This fails to support the idea that vitamin D-sulfate or any other unknown metabolites of vitamin D provide significant vitamin D activity in human milk.