We previously established a mouse neural crest cell line named NCCmelb4, which is positive for Kit and negative for tyrosinase. NCCmelb4 cells were useful to study the effects of extrinsic factors such as retinoic acids and vitamin D(3) on melanocyte differentiation, but in order to study the development of melanocytes from multipotent neural crest cells, cell lines of melanocyte progenitors in earlier developmental stages are needed. In the present study, we established an immortal cell line named NCCmelb4M5 that was derived from NCCmelb4 cells. NCCmelb4M5 cells do not express Kit and are immortal and stable in the absence of Kit ligand. They are positive for melanocyte markers such as tyrosinase-related protein 1 and DOPAchrome tautomerase and they contain stage I melanosomes. Interestingly, glial fibrillary acidic protein, which is a marker for glial cells, is also positive in NCCmelb4M5 cells, while NCCmelb4 cells are negative for this protein. Immunostaining and a cell ELISA assay revealed that 12-O-tetradecanoylphorbol 13-acetate (TPA) and cholera toxin (CT) induce Kit expression in NCCmelb4M5 cells. Real-time polymerase chain reaction analysis also demonstrated the induction of Kit mRNA by TPA and CT. Microphthalmia-associated transcription factor mRNA is simultaneously enhanced by the same treatment. Kit induced by TPA/CT in NCCmelb4M5 cells disappeared after the cells were subcultured and incubated without TPA/CT. These findings show that NCCmelb4M5 cells have the potential to differentiate into Kit-positive melanocyte precursors and may be useful to study mechanisms of development and differentiation of melanocytes in mouse neural crest cells.