In a recent study (Goltz, J.S., Wolkoff, A.W., Novikoff, P.M., Stockert, R.J., and Satir, P. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7026-7030), we found that ligand- and receptor-containing endocytic vesicles bind to endogenous microtubules in vitro after 60 min of receptor-mediated endocytosis of asialo-orosomucoid. In the presence of ATP, ligand-containing endocytic vesicles are released from microtubules, while those containing receptor are not. We hypothesized that cytoplasmic dynein may associate with ligand-containing, but not receptor-containing, domains of endocytic vesicles and might be involved in the movement of ligand-containing vesicles along microtubules during sorting of ligand from receptor. Direct evidence in support of this hypothesis has been obtained in the present study. Binding of ligand-containing vesicles to microtubules correlates highly (p < 0.001) with binding of dynein, but not kinesin, under a variety of conditions. Binding of receptor-containing vesicles to microtubules is independent of both cytoplasmic dynein and kinesin binding. Tight association of cytoplasmic dynein with a population of ligand-containing vesicles is seen directly by immunoprecipitation. These results support the view that in receptor-mediated endocytosis, ligand-containing vesicles become bound to microtubules by cytoplasmic dynein. While receptor domains of endosomes remain attached to microtubules in an ATP-independent manner, ligand-containing domains might be moved away toward pericentrosomal lysosomes by this motor molecule.