AbstractIn situ hybridization, using a biotinylated cDNA probe for the epidermal growth factor receptor(EGFR)gene, indicates that the amplifiedEGFRgenes in the colon tumor cell line, DiFi, are localized in many small double minute chromosomes (dmin) of varying size and visibility. Analysis of the electrophoretic mobility of γ‐irradiated DNA from DiFi by pulsed‐field gel electrophoresis and Southern blot hybridization usingEGFRprobe, indicates that the amplifiedEGFRin DiFi exists in extrachromosomal, covalently closed circular episomes, presumably equivalent to dmin. Two major and one minor species were observed which had estimated sizes of 650, 1,300, and 2,000 kb, respectively. The DiFi cell line appears to represent a unique case of extrachromosomalEGFRgene amplification in human c