We previously showed that plasmids containing both a mammalian replication initiation region and a matrix attachment region were efficiently amplified in human cancer cells and that they were either integrated into preexisting extrachromosomal double minutes (DMs) or induced the generation of a chromosomal homogeneously staining region (HSR). In this article, we elucidated the mechanism by which such plasmids mimic gene amplification. Hybridization experiments using chromatin fiber, metaphase spread, and genomic Southern blot analysis suggested that a circular molecule comprising a plasmid direct repeat was generated initially. Recombination between this molecule and the preexisting DMs led to the apparent stabilization of the plasmid repeat. If the plasmid repeat was integrated into the chromosome, it initiated the breakage-fusion-bridge cycle, which generated HSR. Importantly, we found that HSR formation was blocked by inserting a poly(A) signal or the orientation-specific replication fork barrier downstream of the drug-resistance gene, where the transcription would meet head to head with the supposed replication fork from the initiation region. The matrix attachment region enhanced HSR formation if it was inserted at the same site. These data suggested that strand breakage generated by the conflict between replication and transcription might trigger the breakage-fusion-bridge cycle. This is the first study suggesting that such a conflict leads to genomic instability in higher eukaryotes.