Primary human epidermal melanocytes express six endogenous isoforms of the human actin-associated myosin Va motor protein, involved in organelle transport. As isoforms containing exon F are most abundant in melanocytes, we hypothesized that these isoforms probably have a melanocyte-specific function. To uncover the biologic role of the six isoforms we introduced enhanced green fluorescent protein (eGFP)-myosin Va tail constructs in human melanocytes. We found that the medial tail, undergoing alternative splicing, has to be expressed in combination with the globular tail in order to obtain clear colocalization with organelles. Our data show that isoforms lacking exon F but containing exon D are associated with vesicles near the Golgi area. Myosin Va isoforms containing exon F are able to colocalize with and influence melanosome distribution by indirect interaction with rab27a and direct interaction with melanophilin. These results indicate that the myosin Va medial tail domain provides the globular tail domain with organelle-interacting specificity.