Human thyroxine-binding prealbumin or transthyretin has been isolated by three different methods. In all cases, the isolated material is microheterogeneous in an isoelectric focusing system, revealing a pattern of at least 10 bands. These subforms represent tetramers. Dissociation of the protein in serum samples and dissociation in the presence of urea or sodium dodecyl sulfate (SDS) reveal eight other forms, differing in isoelectric point and concluded to be monomers. Two different sets of dissociated forms are identified, one from urea treatment and the other from SDS treatment. The latter set is apparently also present in serum. Interpretations are complicated by multiplicities of all forms, atypical electrophoretic migrations, nonidentical effects of urea and SDS, and the absence of dimer under most conditions. However, reassociations of monomers and formation of interspecies hybrid molecules identify dimers and clarify interpretations of the dissociated sets. In at least five of the forms likely to represent monomers, the difference is dependent on the nature of the SH group at Cys-10, which can be oxidized, can be reduced, or can be in a mixed disulfide, probably with glutathione and its degradation products. Amino acid sequence analysis reveals an additional N-terminal heterogeneity (with start at positions 1, 2, and 3) of the three most abundant monomers, but this does not explain the observed differences among monomeric forms. It is concluded that prealbumin exhibits different types of microheterogeneity, the major pattern of which is explained by the status of the subunit SH group and the ability of prealbumin to dissociate into monomers. A relation appears to exist between SH group status on the one hand, and retinol-binding protein interaction, the ability to dissociate into monomers, and possibly also binding of thyroxine on the other. These interactions suggest complex functional relationships for prealbumin in serum.