T4-binding globulin (TBG) consists of a single polypeptide chain containing 4 oligosaccharide units with an average of 10 terminal sialic acid residues. Isoelectric focusing (IEF) of TBG purified from pooled human plasma showed 4 main bands isoelectric at pH 4.2, 4.3, 4.5, and 4.6. Removal of 85% of carbohydrates by treatment with mixed glycosidases reduced these bands to 2, at pH 5.4 and 5.7. This residual microheterogeneity was not related to protein-ampholyte complexes, since it was still present in 8 M urea. It also did not represent an equilibrium mixture of interchangeable conformations, since each band obtained in the first dimension IEF gave a single spot when rerun in a second dimension. Therefore, the residual microheterogeneity of TBG after removal of carbohydrates can be attributed to variation in amino acid composition. Since genetic polymorphism of TBG was recently demonstrated, we further investigated whether the residual microheterogeneity was genetically determined. Plasma samples from 20 white donors and 17 black donors were labeled with [125I]T4 and submitted to IEF, followed by autoradiography. TBG-1, found in white donors and most black donors, showed the same 4 bands as TBG purified from pooled plasma. Two less frequent phenotypes were found in black individuals: TBG-2, with 4 bands at approximately pH 4.25, 4.45, 4.55, and 4.7; and TBG-1,2, having all of the bands present in TBG-1 and TBG-2. Electrophoretically homogeneous preparations of TBG of each type were obtained from 100 ml plasma; after deglycosylation, TBG-1 revealed 2 bands isoelectric at pH 5.4 and 5.7, TBG-2 had 2 bands at pH 5.7 and 5.9, and TBG-1,2 had 3 bands at pH 5.4, 5.7, and 5.9. The same TBG bands were found after neuraminidase treatment of whole plasma from the same donors. These data demonstrate two kinds of TBG polymorphism. The first is found in deglycosylated TBG from individual donors and is probably due to variation in amino acid composition. The second, also unrelated to the carbohydrate moiety, is a genetic polymorphism found in blacks.