The concentrations of unconjugated 25-OHD, 24,25(OH)2D, and 1,25(OH)2D were measured in human milk by competitive protein-binding radioassays following successive preparative Sephadex LH-20 chromatography and HPLC. The mean (±SE) concentration of 25-OHD was 0.37±0.03 ng/ml, of 24,25(OH)2D was 24.8±1.9 pg/ml, and of 1,25(OH)2D was 2.2±0.1 pg/ml. The concentration of 25-OHD3 in milk as determined by HPLC and UV detection at 254 nm was 0.27±0.08 ng/ml. The milk concentrations of vitamin D metabolites did not correlate with the maternal serum 25-OHD levels. The total amounts of unconjugated vitamin D metabolites correspond to the known low bioassayable vitamin D antirachitic activity in human milk.