Adaptor protein complexes (APs) are evolutionarily conserved heterotetramers that couple cargo selection to the formation of highly curved membranes during vesicle budding. In Saccharomyces cerevisiae , AP-3 mediates vesicle traffic from the late Golgi to the vacuolar lysosome. The HOPS subunit Vps41 is one of the few proteins reported to have a specific role in AP-3 traffic, yet its function remains undefined. We now show that although the AP-3 δ subunit, Apl5, binds Vps41 directly, this interaction occurs preferentially within the context of the HOPS docking complex. Fluorescence microscopy indicates that Vps41 and other HOPS subunits do not detectably colocalize with AP-3 at the late Golgi or on post-Golgi (Sec7-negative) vesicles. Vps41 and HOPS do, however, transiently colocalize with AP-3 vesicles when these vesicles dock at the vacuole membrane. In cells with mutations in HOPS subunits or the vacuole SNARE Vam3, AP-3 shifts from the cytosol to a membrane fraction. Fluorescence microscopy suggests that this fraction consists of post-Golgi AP-3 vesicles that have failed to dock or fuse at the vacuole membrane. We propose that AP-3 remains associated with budded vesicles, interacts with Vps41 and HOPS upon vesicle docking at the vacuole, and finally dissociates during docking or fusion.