Three charge forms of rat alpha-lactalbumin were separated by ion exchange chromatography on DEAE-cellulose. The amino acid composition of each form was similar but they differed in carbohydrate composition. Each form contained a tryptic glycopeptide having a common polypeptide and heteropolysaccharide unit. The tryptic glycopeptide was sequenced and positioned in rat alpha-lactalbumin, which was partially sequenced from residues 1 to 50. The carbohydrate attachment site was at Asn45. Secondary structure calculations predicted that Asn45 is in a beta bend conformation whereas Asn45 in bovine alpha-lactalbumin, a poorly glycosylated protein, is not in a bend conformation.