Human melanocytes are neural crest-derived cells that synthesize the pigment melanin. These cells migrate from a central location to the dermal-epidermal junction early in gestation and situate themselves between keratinocytes of the basal layer of the epidermis, extending thin dendritic processes upwards into the epidermis. In vitro, neonatal melanocytes can assume a variety of morphologies, depending on the culture conditions. Using standard immunofluorescent, immunoblotting, and Northern blotting techniques, we investigated the expression of intermediate filament proteins and demonstrate here that cultured human melanocytes express vimentin gene and protein under a variety of culture conditions. Vimentin is a 57-kD intermediate filament protein synthesized primarily by cells of mesenchymal origin. It is transcribed as a single-messenger RNA species of 2.0 kb and the human gene is located on chromosome 10. As a member of the intermediate filament family of proteins, we suggest that vimentin is an important component of the cytoskeleton of neonatal, human melanocytes.