Using video intensification fluorescence microscopy and tetramethylrhodamine (Rho)-labeled 3,3',5-triiodo-L-thyronine (T3), we studied the uptake of T3 by cultured mouse fibroblasts. After incubation of cells with Rho-T3 for 30 min at 37 degrees C the fluorescent hormone was concentrated in many small bright accumulations. With a 1000-fold excess of unlabeled T3, only weak background fluorescence was seen. Furthermore, when cells were incubated with Rho or Rho-thyronine only background fluorescence was detected. These results indicate that the cellular uptake of Rho-T3 occurred through a T3-specific receptor-mediated process. Most of these accumulations underwent saltatory motion in living cells, indicating that the T3 was contained within endocytic vesicles. When cultured cells were incubated with Rho-T3 for 60 min at 4 degrees C, only diffuse fluorescence was observed, Rho-T3 became concentrated in vesicles upon warming of the cells to either 23 degrees C or 37 degrees C. Simultaneous incubation of cells with fluorescein-labeled alpha 2-macroglobulin and Rho-T3 showed that Rho-T3 was internalized in the same vesicles as alpha 2-macroglobulin. Furthermore, as previously reported for alpha 2-macroglobulin in the presence of methylamine, dansylcadaverine, or bacitracin, clustering and internalization were inhibited but the overall fluorescence intensity of the cells did not appear to be affected. Because it has been previously shown that receptor-mediated endocytosis of alpha 2-macroglobulin occurs through clustering of ligands in coated pits on the cell surface, these results indicate that Rho-T3 follows the same pathway. Thus it has now been demonstrated that a low-molecular weight hormone enters cells by this pathway.