Thyroid hormone release requires degradation of thyroglobulin (Tg) by thyroid epithelial cells, which occurs mainly in the lysosomal pathway following Tg endocytosis. Non-specific fluid-phase endocytosis is thought to be the main route of Tg uptake leading to degradation, whereas receptor- mediated endocytosis is believed to lead to post-endocytic pathways other than degradation. To gain more insights into these issues, we investigated handling of Tg by various cell types. Tg bound similarly to thyroid (FRTL-5, FRT) and non-thyroid (COS-7, IRPT) cells, indicating the presence of membrane-binding sites, presumably receptors, in both cell types. Tg was internalized and degraded by all cells and degradation paralleled uptake, with the exception of FRTL- 5 cells, in which a lower proportion of Tg was degraded, suggesting that in FRTL-5 cells mechanisms that target Tg to the various post-endocytic pathways (either receptors or postreceptorial factors) are differently represented. Immunoelectronmicroscopy showed a common path of endocytosis in FRTL-5, COS-7, and IRPT cells, namely the formation of pseudopods engulfing Tg, followed by internalization and accumulation of Tg in cytoplasmic vesicles and lysosomes. The fastest rate was observed in COS-7 cells, probably reflecting a lower impact of endocytic receptors. Our findings suggest that Tg uptake and degradation are not thyroid-specific, that Tg binding sites exist in different cell types, and that uptake and/or degradation are differently regulated in differentiated thyroid cells, presumably because of a different impact of endocytic receptors or post-endocytic mechanisms, which are probably responsible for the regulation of hormone release.