A new HPLC procedure is described for the separation and quantitation of minor human Hb variants. The cation exchanger, Synchropak CM 300, is a silica support with a bonded polymeric coating of carboxylic acid residues. The chromatogram is developed with 0.03M Bis-Tris-KCN buffers, pH 6.40, and a Na-acetate gradient increasing from 0 to 0.1125M. As many as 11 minor Hbs (including Hb A2) can be isolated. Some of these have been identified through rechromatography of the minor Hb zones obtained by Bio-Rex-70 chromatography. Quantitation of Hb A1c and Hb A2 is readily accomplished. Hb F0 and an unidentified minor Hb often observed in red cells of alcoholics co-chromatograph with Hb A1c. The method has been applied to blood samples of 10 normal adults, 13 diabetic patients, and nine alcoholic subjects. An excellent correlation exists between the Hb A1c percentages and the levels of Hb A1 determined by microcolumn chromatography. Some other minor Hbs, identified as components 9 and 10, which are (at least in part) Hb A0 with glucose attached to the alpha chains, are present in increased amounts in the blood of diabetic patients and others may be observed in patients who subject themselves to (severe) alcohol abuse. It is suggested that the new procedure is well-suited for detailed studies of the minor Hbs in patients with various abnormalities in carbohydrate metabolism.