We have observed, by Southern blot hybridization, numerous episomes in DNA prepared from tumors grown as athymic mouse xenografts. These extrachromosomal DNAs were present in multiple copies and existed as relaxed and supercoiled conformational isomers. The episomes were readily detected with pBR322 plasmid probes, but not with purified plasmid inserts. Subsequently, four species of bacteria were isolated from tumor xenografts, suggesting that the pBR322 related episomes which we observed were bacterial DNAs, copurified during the isolation of xenograft DNA. This finding illustrates a potential problem which may be encountered in blot hybridizations utilizing nucleic acids from primary tissue preparations.