The variation and distribution of O-glycosidically linked sugar chains in kappa-casein from bovine mature milk were analyzed by HPLC, fast atom bombardment mass spectrometry, and 13C nuclear magnetic resonance. A monosaccharide alditol (N-acetylgalactosaminitol) and four oligosaccharide alditols [a neutral disaccharide (galactosyl-beta 1-3N-acetylgalactosaminitol), two acidic trisaccharides (straight chain: N-acetylneuraminyl alpha 2-3galactosyl-beta 1-3N-acetylgalactosaminitol and branch chain: galactosyl beta 1-3(N-acetylneuraminyl alpha 2-6)N-acetylgalactosaminitol), and an acidic tetrasaccharide(N-acetylneuraminyl alpha 2-3galactosyl-beta 1-3(N-acetylgalactosaminyl alpha 2-6)N-acetylgalactosaminitol] were identified as sugar chains linking on normal bovine kappa-casein. The most dominant sugar chain was an acidic tetrasaccharide. The four oligosaccharide chains were identical to those already found, but N-acetylgalactosamine was a newly identified sugar chain linking on kappa-casein. The distribution of monosaccharide, disaccharide, trisaccharide (straight), trisaccharide (branched), and tetrasaccharide chains were determined by HPLC to be .8, 6.3, 18.4, 18.5, and 56.0%, respectively (means of five kappa-caseins).