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Human and bovine milk were analyzed for vitamin D, 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D, 25,26-dihydroxyvitamin D and 1,25-dihydroxyvitamin D using exhaustive chromatographic purification procedures coupled with ligand binding assays. Human milk contained the following amounts of antirachitic sterols (pg/ml, mean +/- SD, n = 5): 39 +/- 9 vitamin D; 311 +/- 31 25-hydroxyvitamin D; 52 +/- 8 24,25-hydroxyvitamin D; 32 +/- 9 25,26-dihydroxyvitamin D; 5.1 +/- 0.3 1,25-dihydroxyvitamin D. Normal bovine milk contained levels of these sterols comparable to those found in human milk. Increasing the oral dose of vitamin D to the cows was reflected by an increase of the parent vitamin and 25-hydroxyvitamin D in the milk. Vitamin D-binding protein concentration in human milk whey, determined by Ouchterlony immunodiffusion and radioimmunoassay, was 1--2% of the levels observed in the plasma and was dependent on the stage of lactation. Vitamin D and its metabolites were shown initially to be present in the whey portion but with time migrated into the fat portion of milk. The antirachitic sterols detected account for approximately 25 IU/liter and 27 IU/liter of antirachitic activity in human and bovine milk, respectively. In both species 25-hydroxyvitamin D comprised the majority of the antirachitic sterols detected in normal milk.