Yeast trans-Golgi network (TGN) membrane proteins maintain steady-state localization by constantly cycling to and from endosomes. In this study, we examined the trafficking itinerary and molecular requirements for delivery of a model TGN protein A(F-->A)-alkaline phosphatase (ALP) to the prevacuolar/endosomal compartment (PVC). A(F-->A)-ALP was found to reach the PVC via early endosomes (EEs) with a half-time of approximately 60 min. Delivery of A(F-->A)-ALP to the PVC was not dependent on either the GGA or adaptor protein 1 (AP-1) type of clathrin adaptors, which are thought to function in TGN to PVC and TGN to EE transport, respectively. Surprisingly, in cells lacking the function of both GGA and AP-1 adaptors, A(F-->A)-ALP transport to the PVC was dramatically accelerated. A 12-residue cytosolic domain motif of A(F-->A)-ALP was found to mediate direct binding to AP-1 and was sufficient to slow TGN-->EE-->PVC trafficking. These results suggest a model in which this novel sorting signal targets A(F-->A)-ALP into clathrin/AP-1 vesicles at the EE for retrieval back to the TGN.