Amplification of the human N-MYC proto-oncogene is frequently seen either in extrachromosomal double minutes or in homogeneously staining regions of aggressively growing neuroblastomas. N-MYC maps to chromosome 2 band p23-24, but homogeneously staining regions have never been observed at this band, suggesting transposition of N-MYC during amplification. Previous studies had suggested that in cells with amplified N-MYC the chromosomes 2 appear to be unaltered and to carry one apparently normal copy of N-MYC each. In contrast, the contribution of N-MYC to tumors which lack amplification has been unclear. We here show, by fluorescence in situ hybridization, that N-MYC is occasionally duplicated at its resident site in neuroblastoma cell lines previously thought to have a single copy gene. Additionally, we detected duplication in a neuroblastoma cell line carrying amplification. Our results raise the possibility that duplication may, in some neuroblastomas, either be a prelude to amplification or an alternative pathway by which N-MYC becomes activated.