Purification of bovine thyrotropin and lutropin by conventional soft-gel chromatographic methods is shown to yield microheterogeneous products. High-performance ion-exchange chromatography (HPIEC) can be used to purify these isohormones further for investigations into the structure/function role of glycoprotein microheterogeneity. Hormonal microheterogeneity is shown to be reflected in differences in surface-accessible charged groups, as demonstrated by HPIEC, and differences in net charge, as indicated by isoelectric focusing. Optimal separation of the glycoprotein hormones, based on protein charge, can therefore be achieved by a combination of these two methods.