This study describes characteristics of two newly established cell lines (OSC-1 and OSC-2), derived from two oesophageal squamous cell carcinomas. Morphologically, OSC-1 cells and OSC-2 cells grew in epithelial cobblestone patterns with cells piling up to 4 cells. Ultrastructurally, both cell lines showed formation of desmosomes; however, tonofilaments were only formed by OSC-2 cells. Immunohistochemical investigations revealed coexpression of vimentin and cytokeratin in OSC-1 cells and OSC-2 cells. A cytokeratin subtype typical for mature squamous epithelia (cytokeratin 13) was expressed only in OSC-2 cells. OSC-1 cells showed tumour formation in nude mice, whereas OSC-2 cells did not. Cytogenetic analysis revealed that OSC-1 cells had a hyperdiploid karyotype and OSC-2 cells had a near-triploid karyotype. In both cell lines, the formation of multicellular spheroids could be induced. In conclusion, in comparison with OSC-2 cells the OSC-1 cells were characterized by a poorer degree of differentiation and by a more aggressive growth behaviour in vivo.