Cultured monkey hepatocarcinoma cell (NCLP-6E) were used to investigate the uptake and metabolism of thyroid hormones. Intracellular accumulation was shown by the failure to acutely release hormone from cells subsequently exposed to serum proteins, and by the metabolic trnasformation of the hormones to deiodinated products and their sulfates. When hepatocarcinoma cell monolayers were studied at hormone concentrations below 10(-10) M, neither KCN nor dinitrophenol inhibited uptake. Taken together with previous findings that uptake was neither saturable nor reduced at low temperature, these results indicate that this process was not active transport. Deiodination of both the phenolic and non-phenolic rings, however, was partially inhibited by KCN but not by dinitrophenol. Sulfation of 3,3'-diiodothyronine and 3'-monoiodothyronine was strongly inhibited by both KCN and dinitrophenol. Uptake of the hormones and their metabolites was also measured in suspended hepatocarcinoma cells and compared with the uptake by normal rat hepatocytes, human fibroblasts and human lymphocytes. In these experiments 1 micrometer triidothyronine and 0.47 mM dinitrophenol were used to inhibit deiodination and sulfation, respectively. Uptake was similar in all cell types. Accumulation was highest with 3,5,3'-triiodothyronine, intermediate with other compounds having iodines in both rings, lowest with compounds iodinated in only one ring, and absent with iodothronine sulfates. These findings help to explain the relative rates of metabolism of the iodothyronines and their release from the cells.