The cytoplasmic domain of transferrin receptor (TR) is essential for endocytosis of this transmembrane protein. We have investigated by electron microscopy the association of wild-type and cytoplasmic deletion mutant human TR with coated pits at the surface of transfected L cell lines. Approximately 15% of wild-type TR was concentrated in coated pits, regardless of the level of TR expression. In contrast, only 2% of deletion mutant TR was present in these structures. We also correlated the frequency of coated pits with the level of TR expression in different transfected L cell lines. Expression of more than 3 x 10(6) wild-type TR per cell was accompanied by up to a 4-fold increase in coated pits compared with nontransfected Ltk- cells. No such increase was observed in a cell line expressing a similarly high level of cytoplasmic deletion mutant TR. These results indicate that the cytoplasmic domain plays an active role in sorting and endocytosis of TR by providing an assembly site for coated pit formation.