The cell line B95-8 releases Epstein-Barr virus (EBV) with high titres of transforming activity and is widely used as a model in cancer research and virology. There are, however, controversial reports about the species of origin, cell line stability and karyotype. To address these questions, B95-8 chromosomes were analysed by chromosome sorting and painting by multicolour fluorescence in-situ hybridization. Reciprocal painting was performed between B95-8, 'wildtype' New World monkey and human chromosomes. Saguinus oedipus was revealed as the species of origin. A further five cell-line-specific marker chromosomes, resulting from translocations, deletions and an insertion were found. Although human chromosome 6 or 13 homologues were always involved in these rearrangements, co-hybridization of an EBV-specific DNA probe did not reveal site-specific hybridization to marker chromosomes or at translocation breakpoints. The multicolour probe set described here will be of special value for further evolutionary studies in New World monkeys.