In this liquid-chromatographic assay for acetylated hemoglobin in human blood, glycated hemoglobins in hemolysates are first removed by affinity chromatography on boronate-agarose columns. Acetylated hemoglobin in the nonretained fraction is determined by cation-exchange chromatography. The absorbance of the effluent is monitored at 415 nm. The mean within-assay CV was 8.5%, the between-assay CV 17%. The mean proportion of acetylated hemoglobin in 20 pregnant, nondiabetic women was 1.9%, and in 17 alcoholics it was 2.7%. Rapid and reproducible, this method is suitable for use in routine determination of acetylated hemoglobin.