ZW10 interacts with dynamitin, a subunit of the dynein accessory complex dynactin, and functions in termination of the spindle checkpoint during mitosis and in membrane transport between the endoplasmic reticulum (ER) and Golgi apparatus during interphase. Its associations with kinetochores and ER membranes are mediated by Zwint-1 and RINT-1, respectively. A previous yeast two-hybrid study showed that the C-terminal region of ZW10 interacts with dynamitin, and part of this region has been used as an inhibitor of ZW10 function. In the present study, we reinvestigated the interaction between ZW10 and dynamitin, and showed that the N-terminal region of ZW10 is the major binding site for dynamitin and, like full-length ZW10, could potentially move along microtubules to the centrosomal area in a dynein-dynactin-dependent manner. Competitive binding experiments demonstrated that dynamitin and RINT-1 occupy the same N-terminal region of ZW10 in a mutually exclusive fashion. Consistent with this, over-expression of RINT-1 interfered with the dynein-dynactin-mediated movement of ZW10 to the centrosomal area. Given that the N-terminal region of ZW10 also interacts with Zwint-1, this region may be important for switching partners; one partner is a determinant for localization (kinetochore and ER) and the other links ZW10 to dynein function.