OBJECTIVE - To directly compare results obtained using an ion-exchange high-performance liquid chromatography (HPLC) HbA(1c) method used in the Diabetes Control and Complications Trial with two different affinity chromatography methods in which 'total GHb' is determined. RESEARCH DESIGN AND METHODS - Blood was obtained from a large number of people with and without known diabetes. The specimens were divided and assayed for HbA(1c) and for total GHb. Total GHb was determined using a semi-automated gravity- elution boronate affinity chromatography method and an automated boronate affinity HPLC method. The results obtained with the two methods were also compared. RESULTS - In subjects without known diabetes, the mean percentage HbA(1c) and the range of values were similar to the total GHb values in the same subjects when assayed using the semi-automated affinity gravity-elution method (mean 5.2 ± 0.4 and 5.1 ± 0.4% [SD], respectively). With the affinity HPLC method, results were 5.3 ± 0.4%. The similarity in results was surprising. However, analysis of the data suggests that a large proportion of the material in the HbA(1c) fraction measured using this ion-exchange HPLC method is not GHb, as pointed out by others. Although the results were similar in people without known diabetes, in the people with diabetes, the incremental increase was ~25% greater for the total GHb when compared with the increase in HbA(1c). When corrected for the non-GHb being measured by the HbA(1c) method, it can be calculated that ~40% more GHb is measured using affinity chromatography over the entire range of GHb values. CONCLUSIONS - The similarity in the mean and range of percent HbA(1c) and in percent total GHb using these different methods can be attributed to two factors: 1) the HbA(1c) ion-exchange method measures only ~50-60% of the total GHb present, and 2) ~40-50% of the material being measured in the HbA(1c) fraction is not GHb, i.e., offsetting factors fortuitously resulted in values similar to the more specific affinity methods. The greater incremental increase in percent total GHb compared with percent HbA(1c) in people with diabetes can be attributed to the greater amount of GHb being measured with the affinity methods.