Glucose spontaneously reacts with hemoglobin amino groups to produce unstable Schiff base complexes that can dissociate or rearrange to form stable Amadori products. We used dynamic capillary isoelectric focusing and boronate affinity chromatography to assess the formation and dissociation of unstable hemoglobin complexes in vitro. Formation was studied by incubating erythrocytes at 37°C for up to 24h in phosphate-buffered saline (PBS) supplemented with 0 to 55.6 mmol/L glucose. Dissociation was studied by incubating glucose-loaded erythrocytes in PBS without glucose. Dynamic capillary isoelectric focusing separated hemoglobin A1c into two subfractions identified as A1c1 and A1c2. The A1c1 subfraction contained both stable and unstable hemoglobin complexes. The A1c2 subfraction contained only unstable hemoglobin complexes. Both subfractions quantitatively increased in the presence of glucose and decreased in its absence. Rates of increase and decrease were faster and time to equilibrium was shorter for A1c2 (~4 h) compared with A1c1 (~20 h). Unstable hemoglobin complexes did not bind to boronate affinity columns but instead eluted intact in A1c1 and A1c2 subfractions from nonglycated affinity fractions. Cyanoborohydride reduction confirmed the presence of Schiff base complexes. Evidence of multiple unstable hemoglobin complexes with different rates of glycation suggests that new models are needed to describe nonenzymatic hemoglobin glycation.