Isoelectric focusing (IEF) of human erythrocyte hemolystaes on polyacrylamide slab gels over a pH gradient of 6 to 8 provides sufficient resolution of hemoglobin AIc (HbAIc) from other hemoglobin components (AIa, AIb, AII, S, and F) to allow quantification by high-resolution microdensitometry. Twice-chromatographed HbAIc alone and in admixtures with normal and diabetic hemolysates were employed to verify the identification and quantification of the AIc component. Relative concentration was determined as a per cent of the total hemoglobin absorption at 556 nm on acid-fixed, unstained gels. A total of 60 patient samples were examined by IEF, and, for 35 samples, both column chromatography and IEF determination were obtained, revealing excellent correlation between these two methods.