The intracellular fate of radiolabeled T3 taken up by mice hepatocytes in vivo was determined at specific time intervals (2-120 min) after injection by quantitative electron microscopic radioautography. Injection of a 200-fold excess of unlabeled T3 together with [125I]-T3 resulted in a more than 90% inhibition of radioactivity detected in hepatocytes. A simple grain density (GD) analysis of radioautograms revealed that a specific labeling (GD > 1) was displayed by only five cell compartments: the plasma membrane, lipid droplets, mitochondria, nuclear envelope and nuclear matrix whereas other compartments were not labeled. Labeled compartments showed distinct changes in the pattern of labeling over time: the plasma membrane was labeled only 2 min after T3 injection, whereas labeling of the nuclear envelope was high at 2 min, decreased at 15 min and progressively increased to maximal measured levels at 120 min. After a lag time of 30 min, nuclear matrix labeling increased progressively with time. Mitochondrial labeling was found to be specific at any time point studied but showed no change over time. These ultrastructural data have been confirmed in vitro by the interaction of T3 with plasma membrane, nuclear membrane, nuclear matrix and mitochondria by real-time biospecific interaction analysis in a BIAcore system. These results demonstrate that T3 binds to hepatocytes before internalization, is transported both to mitochondria and to the nuclear envelope and translocated into the nuclear matrix.