Amplified MYCN, common in neuroblastomas, can be detected as double minutes (dmin) or homogenously staining chromosomal regions (hsr). Expulsion of amplified MYCN has only been described in dmin. We used hydroxyurea (HU), which accelerates the expulsion of amplified genes and cytostatics (used in neuroblastoma therapy), to describe MYCN amplification changes after chemotherapy. We used IMR-32, SK-N-AS, UKF-NB-2, UKF-NB-3, UKF-NB-4, and derived sublines resistant to doxorubicin, cisplatin, and vincristine. The loss of amplified MYCN copies was investigated using comparative genomic hybridization and by fluorescent in situ hybridization. We found expulsion of amplified MYCN from hsr in UKF-NB-4 and IMR-32 cell lines, and determined the exact number of amplified MYCN copies. After the first cultivation with HU, some amplified MYCN was lost. UKF-NB-4 lost 20 copies on average, and IMR-32 lost 15 copies (P<0.001). After the second cultivation, cells without MYCN amplification were found. In comparison to sensitive cell lines, drug-resistant cell lines lost 17 copies on average. Our data show that expulsion of amplified MYCN genes is also possible from hsr and may be induced, not only by HU, but by other cytostatics as well.