DNA amplification is an important mechanism of tumor progression that allows cancer cells to up-regulate the expression of critical genes such as oncogenes and genes conferring drug resistance. Recent studies using comparative genomic hybridization (CGH) revealed increased DNA copies of 20q sequences in 7 melanoma cell lines and B archival metastatic melanoma lesions. To evaluate chromosome 20 abnormalities in more detail and to resolve discrepancies between karyotype and CGH findings, we performed FISH analysis of metaphase cells in 13 melanoma cell lines (including the 7 lines used for CGH) and 9 primary melanoma specimens by using a whole chromosome paint specific for chromosome 20. All 13 cell lines (100%) and 8/9 primary tumors (89%) showed extra copies of chromosome 20 relative to tumor ploidy. Additionally, 6/14 cell lines (43%) and 2/8 primary tumors (25%) showed translocated chromosome 20 material previously undetected by standard cytogenetics. Cytologic evidence for gene amplification was also found in one cell line, which contained an add(20)(p13), with additional DNA being derived from 20q sequences. These data suggest that overrepresentation of a gene or genes important for melanoma pathogenesis resides on the long arm of chromosome 20.