Rab15 is a novel endocytic Rab that counters the stimulatory effect of Rab5-GTP on early endocytic trafficking. Rab15 may interfere with Rab5 function directly by sequestering Rab5 effectors or indirectly through novel sets of effector interactions. To distinguish between these possibilities, we examined the effector binding properties of Rab15. Rab15 does not interact directly with the Rab5 effectors rabex-5 and rabaptin-5 in a yeast two-hybrid binding assay. Rather mammalian suppressor of Sec4 (Mss4) was identified as a binding partner for Rab15. Mss4 preferentially binds GDP-bound (T22N) and nucleotide-free (N121I) Rab15, consistent with the proposed role of Mss4 as a chaperone that stabilizes target Rabs in their nucleotide-free form. Mutational analysis of Rab15 indicates that lysine at position 48 (K48Q) is important for the binding of Rab15-GDP to Mss4. Moreover, the mutation K48Q counters the inhibitory phenotype of wild type Rab15 on receptor-mediated endocytosis in HeLa cells and homotypic endosome fusion in vitro without altering the relative amount of cell surface-associated transferrin receptor. Together, these data indicate a novel role for Mss4 as an effector for Rab15 in early endocytic trafficking.