We have utilized a dihydrofolate reductase (DHFR) probe in combination with selected probes from other positions along the 2q chromosome arm in a two-color fluorescence in situ hybridization analysis of early DHFR gene amplification events in CHO cells. These studies show clearly that the most frequent initiating event is the formation of a giant inverted duplication, resulting from chromosome breakage and terminal fusion or a reverse unequal sister chromatid exchange. The dicentric chromosomes thus formed initiate bridge/breakage/fusion cycles that appear to mediate subsequent amplification steps to higher copy number.