Hemoglobin AIc is a minor component of normal adult erythrocytes whose concentration is elevated approximately 2-fold in patients with diabetes mellitus. Previous work suggested that the unique structural feature of hemoglobin AIc is the presence of a low molecular weight sugar moiety at the NH2-terminal valine of the beta chain. In this study the structure of the carbohydrate moiety and the nature of its linkage of the beta chain were investigated. Enzymatic digestion of borohydride-reduced betaAIc chains followed by ion exchange chromatography led to the isolation of two distinct NH2-terminal glycovalylhistidines. Comparison of these glycodipeptides with synthetic glycovalylhistidines by thin layer chromatography, gas-liquid chromatography, and proton magnetic resonance spectroscopy gave direct evidence that the naturally derived materials correspond to glucitol and mannitol valylhistidines. Model reactions showed that glucose and mannose react with valine under mild conditions to form an adduct which upon sodium borohydride reduction yields in both cases glucitol and mannitol valines. This suggests a common intermediate, 1-deoxy-1-(N-valyl)fructose, for both reactions. From these studies we conclude that hemoglobin AIc has, as the NH2 terminus of the beta chain, 1-deoxy-1-(N-valyl)fructose. The possible biosynthetic pathways of hemoglobin AIc are discussed.