Thyroxine-binding prealbumin (TBPA) and thyroxine-binding globulin (TBG) were isolated from canine serum and partially characterized. TBPA was isolated by retinol-binding protein (RBP) affinity chromatography and further purified by preparative agarose gel electrophoresis or FPLC ion exchange chromatography. TBG was purified by thyroxine (T4)-Sepharose chromatography followed by gel filtration on Sephacryl S-300 and preparative electrofocusing in a granulated dextran gel. Molecular weights were estimated by SDS-polyacrylamide gradient gel electrophoresis. Canine TBPA had a tetramer molecular weight of 56,000, an extinction coefficient of 12.8 cm2cg.1, an isoelectric point of 5.26-5.70 and a microheterogeneity pattern similar to that of human TBPA. Partial immunochemical identity with human TBPA was also found. Plasma concentrations of TBPA were measured by rocket immunoelectrophoresis in 43 normal and 35 hypothyroid dogs. Reference levels for TBPA ranged between 205 and 474 mg/l. Hypothyroid dogs had a mean TBPA level of 315.0 mg/l (SD: 91.1 mg/l). TBG had a molecular weight of 75,000 and an isoelectric point of 5.0. No immunochemical identity with human TBG was found. Gel filtration of serum on Sephacryl S-200, identification of T4-binding proteins with 125I-T4, and protein- and lipoprotein staining of fractions was performed. Thyroxine-binding was found to TBG in the beta-globulin region, TBPA in the alpha 2-region, albumin, and to the high density lipoprotein (HDL2) in the alpha 1-region and the very low density lipoprotein (VLDL) in the pre-beta region. A corresponding band to the latter protein in serum was masked by TBG and TBPA, and T4-binding in the alpha 1-region was not always seen in serum. Many similarities were found between man and dog regarding TBPA, but not TBG. The differences in structure of TBG may in part be responsible for the low serum T4 levels and rapid T4 metabolism seen in dogs.