ABSTRACT: The maturation of organelles in the endolysosomal pathway requires the exchange of the early endosomal GTPase Rab5/Vps21 for the late endosomal Rab7/Ypt7. The Rab exchange depends on the GEF activity of the Mon1-Ccz1 heterodimer for Ypt7. Here, we investigated the vacuole binding and recycling of Mon1-Ccz1. We found that Mon1-Ccz1 was absent on vacuoles lacking the phosphatidic acid phosphatase Pah1, which also lack Ypt7, the phosphatidylinositol 3-kinase Vps34 and the lipid PI3P. The interaction of Mon1-Ccz1 with wild-type vacuoles required PI3P as shown in competition experiments. We also found that Mon1 was released from vacuoles during the fusion reaction and its release required its phosphorylation by the casein kinase Yck3. In contrast, Mon1 was retained on vacuoles lacking Yck3 or when Mon1 phosphorylation sites were mutated. The phosphorylation and release of Mon1 was restored with the addition of recombinant Yck3. Together this study showed that Mon1 is recruited to endosomes and vacuoles by PI3P, and - likely after activating Ypt7 - is phosphorylated and released from vacuoles for recycling.