The identities and relative amounts of the major metabolites in rat thyroids 6 h after the administration of [14C]propylthiouracil ([14C]PTU) have been investigated. Rat thyroid extracts were chromatographed on columns of Bio-Gel P-2 and DEAE-Sephadex and in various thin layer chromatography systems. The extracts contained protein-bound PTU metabolites; an unknown peak 3; peak 1, which was chromatographically similar to PTU--SO2H; peak 2, which was similar to PTU--SO3H; PTU; and small amounts of 6-n-propyluracil (PU). The major metabolites were isolated and purified by column chromatograpy. On the basis of chromatographic properties identical to cochromatographed standards in seven different systems and the products formed after treatment with various reagents, peak 1 was identified as PTU-SO2H and peak 2 as PTU--SO3H. Peak 3 was seen only on Bio-Gel P-2 columns, was very unstable, and was not similar to any known PTU standard. The properties of this compound suggest that it may be a thiolsulfonic ester (formula:see text), but the data are insufficient for positive identification. Approximately 85% of the radioactivity in the protein peak was bound to thyroglobulin. HCl converted 86.5% of the protein-bound radioactivity to PU, and H2S converted 91% to PTU, indicating that an oxidized S was involved in the linkage to protein. Dithiothreitol released 23.6% of the protein-bound radioactivity as PTU, and mercaptoethanol released 32.5%, indicating that 25-35% of the PTU is bound in disulfide linkage. Approximately 50% of the radioactivity released by mercaptoethanol was S-ethanol PTU, which suggests a PTU-protein bond similar to a thiolsulfonic ester. Quantitation of the metabolites revealed that protein-bound metabolites accounted for 21-29% of the total radioactivity, unknown peak 3 accounted for 7.1%, PTU--SO2H for 48-50%, PTU--SO3H for 8-10%, and PTU for 10.7-16.5%. Only traces of PU were observed. The data demonstrate that PTU--SO2H is the major PTU metabolite in rat thyroid and must be the compound X observed by other investigators and that all metabolites identified are oxidative products of PTU. These findings support the earlier conclusion of Taurog and Riesco that protein binding of PTU occurs as a consequence of oxidation.