By affinity chromatography on concanavalin A (ConA) linked to Sepharose, S-carboxymethylated rat alpha-fetoprotein could be separated into two molecular variants: a ConA-reactive and a ConA-nonreactive fraction. The carbohydrate chains were quantitatively released from the protein by hydrazinolysis. Based on methylation analysis and high-resolution 1H-NMR spectroscopy of the re-N-acetylated hydrazinolysates, the carbohydrate structures of the two ConA-molecular variants of alpha-fetoprotein were established. The ConA-reactive species contains two N-glycosidic carbohydrate units per molecule, both having the following structure: (formula; see text) The ConA-nonreactive species possesses also two N-glycosidically linked oligosaccharide chains per molecule; each of these has the following structure: (formula; see text)