The contents of total, free, and bound vitamin B₂ (B₂) in bovine milk and their distribution in four separate milk fractions, including milk during the early lactation stage, were estimated. The total B₂ content in whole mature milk was 179±25μg/100g (n=16), and its distribution in the cream, whey, skim milk membrane, and casein fractions was 6, 67, 9, and 18%, respectively. The amount of flavins bound to protein in the total B₂ was 13.6% in whole milk and rich in membrane fraction. The total B₂ content (μg/100g of milk) was higher in colostrum at 1-3 days (287±120) than in colostrum at 4-7 days (173±27), in transitional milk (182±33), and in mature milk (179±44). The bound flavin content decreased slightly as lactation progressed (20-30μg/100g), but the ratio of bound/total B2 did not vary (12-15%). Milk fat globule membrane (MFGM) contained 414±65μg of B₂/g of protein, most of it being bound to protein (92%). Market milks contained as much total B₂ as raw whole milk, but the amount of bound form was only 2%. Guanidine HCl, urea, sodium dodecyl sulfate, pH at 3.0-3.5, delipidation, and boiling released most of the B₂ bound to protein, suggesting that bound flavins bind to milk proteins by a hydrophobic linkage.