Purified human thyroglobulin (Tg) was enzymatically digested with trypsin. After completion of digestion, the tryptic digest was fractionated by gel filtration on a Biogel A 1.5 m column. Further separation and isolation of the major peak C was carried out on a Sephadex G-75 column. Nine fractions were separated and antigenic properties evaluated by a specific and sensitive radioimmunoassay using a rabbit antiserum to 19S Tg and three different antisera from patients with autoimmune thyroid diseases. The Tg fragments react with both hetero- and auto-antisera. The highest antigenic activity was found on larger fragments, but a fairly good activity was also observed on fragments (C6, C7) with lower molecular weight. Antigenic determinants of Tg differ in individual sera suggesting that different sets of determinants elicit the autoimmune process.