Canine thyroglobulin (cTg) has been isolated and purified. It has similar electrophoretic patterns as Tg from other mammalian species. The main fraction had a MW of 660,000, whereas also fractions of a MW of approximately 1,300,000 (dimer) and 330,000 (subunit) were present. The iodine content was 0.8 to 1.0% (w/w). cTg did not cross-react with antibodies against human Tg to a degree that would allow the use of a radioimmunoassay for human Tg for the determination of cTg in serum or plasma. Therefore a polyclonal antiserum was raised against cTg and a homologous radioimmunoassay was developed, which was sensitive (0.4 micrograms/l) and specific (cross-reactivity in cTg assay of human Tg, goat Tg, T4, T3, and DIT less than 0.01%). Plasma Tg levels in normal dogs of both sexes and aged 3-15 years amounted to 192 +/- 73 micrograms/l (mean +/- SD, n = 30). There was no relation between plasma Tg and T4 levels.