Amplification of the N-myc oncogene is detected in about 30% of untreated neuroblastomas. Amplification is associated with advanced stages of disease and rapid tumor progression. However, it was not known if the N-myc copy number was homogeneous in tumor tissue of an individual patient, or if it changed with time in vivo. Therefore, we have made 66 observations on multiple simultaneous or consecutive tumor samples from 60 patients with neuroblastoma. (a) Simultaneous samples were obtained from different areas of 31 tumor masses from 30 patients: a similar N-myc copy number (1-2, 3-10, or greater than 10) was found in all samples from each patient. (b) Simultaneous samples were obtained from different anatomical sites in ten patients. No difference in N-myc copy number was seen. (c) Finally, 25 patients had two or more tumor samples obtained over time. Thirteen patients had a single copy of N-myc in all samples, and 12 had consistent levels of amplification in all samples. Two of the latter cases had single copy of N-myc in a second-look surgery sample, but no tumor was evident histologically. This study demonstrates that the N-myc copy number in human neuroblastomas is usually consistent within a tumor, not only at different tumor sites, but also at different times in vivo. Overall, these findings suggest that N-myc amplification is an intrinsic biological property of a subset of neuroblastomas, and if amplification is going to occur, it is generally present at the time of diagnosis.