Rat alpha-lactalbumin is unique in contrast to alpha-lactalbumin isolated from other species in that it exists in three charge forms. Each form contains carbohydrate and is active in the lactose synthetase reaction. Form II comprises about 80% of the total alpha-lactalbumin and contains a single heteropolysaccharide unit which is attached to the polypeptide chain at Asn45. The detailed structure of this unit was ascertained using specific exoglycosidases, endoglycosidases, and methylation analysis. The following structure is proposed for the heteropolysaccharide unit: (formula: see text).