The expression and degree of amplification of the MYCN oncogene in neuroblastoma provide an important indicator of disease prognosis. Detection of MYCN amplification has been described using Southern blotting or polymerase chain reaction (PCR) on DNA from fresh or frozen tissue samples, and using in situ hybridization mainly on metaphase spreads or smears of cultured neuroblastoma cells. In this article, we describe fluorescence in situ hybridization (FISH) results on detection of MYCN amplification in formalin-fixed, paraffin-embedded samples of 25 neuroblastoma and 20 nonneuroblastoma pediatric tumors. MYCN amplification was readily detectable by FISH in eight of the neuroblastomas; correlation with results obtained by Southern analysis was perfect. Of the nonneuroblastoma tumors, only one of three retinoblastoma cases showed MYCN amplification. In contrast to the Southern blot technique, FISH demonstrated the state of amplification heterogeneity of the tumor cells as well as the nature of the amplification units: double-minute chromosomes (DMs) or homogeneously staining regions (HSRs). The results indicate that FISH is a rapid and reliable method for detection of MYCN oncogene amplification in routinely processed samples and may be used to supplant the Southern blot technique.