The metabolism of thyroxine, 3,3'-triiodothyronine and 3,3',5'-triiodothyronine was investigated in rat hepatoma cell cultures (R117-21B). These iodothyronines were labeled with 125I in the phenolic ring and the metabolites were analyzed by ion-exchange column chromatography. When thyroxine was incubated with the cells at 37 degrees C, its glucuronide was the major product and a little increase in 125I- was detected. Although 3,3',5-triiodothyronine was not observed in the incubation medium, this metabolite was clearly identified in the ethanol extract obtained from the cell homogenates after 24 h incubation. This cell line also metabolized labeled 3,3',5-triiodothyronine added to culture medium. After 24 h incubation, 3,3',5-triiodothyronine glucuronide was the major metabolite and iodothyronine sulfates were also formed. The sulfates contained, 3,3',5-triiodothyronine and 3,3'-diiodothyronine sulfates and an unknown component. In the metabolism of 3,3',5'-triiodothyronine, the cells were very active in carrying out glucuronidation and phenolic ring deiodination, and this metabolism yielded 3,3',5'-triiodothyronine and 3,3'-diiodothyronine glucuronides. The iodide fraction contained a small amount of 3,3'-diiodothyronine sulfate. These results show that the R117-21B rat hepatoma cells metabolize the thyroid hormones and their analogs by phenolic and nonphenolic ring deiodinations, by glucuronidation and by sulfation.