The well-known coat-color mutant mouse dilute exhibits a defect in melanosome transport, and although various mutations in the myosin-Va gene, which encodes an actin-based motor protein, have been identified in dilute mice, why missense mutations in the globular tail of myosin-Va, a putative cargo-binding site, cause the dilute phenotype (i.e. lighter coat color) has never been elucidated. In this study we discovered that missense mutations (I1510N, M1513K and D1519G) in the globular tail (GT) of myosin-Va partially impair the binding of Slac2-a/melanophilin, a linker protein between myosin-Va and Rab27A on the melanosome. The myosin-Va-GT-binding site in Slac2-a was mapped to the region (amino acids 147-240) adjacent to the N-terminal Rab27A-binding site, but it is distinct from the myosin-Va-exon-F-binding site (amino acids 320-406). The myosin-Va-GT.Slac2-a interaction was much weaker than the myosin-Va-exon-F.Slac2-a interaction. The missense mutations in the GT found in dilute mice abrogated only the myosin-Va-GT.Slac2-a interaction and had no effect on the myosin-Va-exon-F.Slac2-a interaction. We further showed that expression of green fluorescence protein-tagged Slac2-a lacking the myosin-Va-GT-binding site (DeltaGT), but not the wild-type Slac2-a, severely inhibits melanosome transport in melan-a cells, especially at the melanosome transfer step from microtubles to actin filaments (i.e. perinuclear aggregation of melanosomes). On the basis of our findings, we propose that myosin-Va interacts with Slac2-a.Rab27A complex on the melanosome via two distinct domains, both of which are essential for melanosome transport in melanocytes.