Gene amplification is frequently associated with tumor progression, hence, understanding the underlying mechanisms is important. The study of in vitro model systems indicated that different initial mechanisms accumulate amplified copies within the chromosomes (hsr) or on extra-chromosomal elements (dmin). It has long been suggested that formation of dmin could also occur following hsr breakdown. In order to check this hypothesis, we developed an approach based on the properties of the I-SceI meganuclease, which induces targeted DNA double-strand breaks. A clone containing an I-SceI site, integrated by chance close to an endogenous dhfr gene locus, was used to select for methotrexate resistant mutants. We recovered clones in which the I-SceI site was passively co-amplified with the dhfr gene within the same hsr. We show that I-SceI-induced hsr breakdown leads to the formation of dmin and creates different types of chromosomal rearrangements, including inversions. This demonstrates, for the first time, a direct relationship between double-strand breaks and inversions. Finally, we show that activation of fragile sites by aphidicolin or hypoxia in hsr-containing cells also generates dmin and a variety of chromosomal rearrangements. This may constitute a valuable model to study the consequences of breaks induced in hsr of cancer cells in vivo.