To investigate characteristics of malignant melanomas with various pathobiological features, a homotransplantable tumor line (RMM) was established from a spontaneous amelanotic melanoma found in the pinna of an aged F344 rat. RMM tumors were transplanted in syngeneic rats by serial subcutaneous implantation with 100% intake. The original and RMM tumors consisted of spindle-shaped cells arranged mainly in interlacing bundles. Immunohistochemically, the neoplastic cells were positive to PNL-2 (melanocytes), nestin (neuroectodermal stem cells), S-100 (neurogenic cells) and vimentin (mesenchymal cells). Electron microscopically, tumor cells possessed single membrane-bound pre-melanosomes. Further, a cell line (RMM-C) was induced from an RMM tumor. RMM-C cells and the induced tumors in syngeneic rats showed immunohistochemical reactions similar to the original and RMM tumors. Interestingly, serum level of galectin-3 expression was increased with growing RMM tumors, and the expression was influenced by TNF-α (increase) or TGF-β1 (decrease), indicating a possible biomarker of amelanotic melanomas. The RMM tumors and RMM-C cell line could become useful tools for studies on the pathobiology, including tumor immunity, and development of therapeutic strategies against this malignancy. These tools are the first tumor lines of amelanotic melanomas in the rat.