Adenovirus type 12 induces four fragile sites upon infection of human cells. The U2 locus, consisting of up to 20 tandem repeats of a 5.8-kbp monomer, maps at the most sensitive of these sites at 17q21-22. We have previously shown that an artificial U2 locus integrated into the human genome generates a new virus-induced fragile site. To determine which elements within the U2 monomer are responsible for fragility, we constructed loci consisting of tandem repeats of subfragments of the U2 monomer. With this approach, we demonstrate that a transcriptionally competent U2 gene is necessary and sufficient for virus-induced fragility and that no other element within the 5.8-kbp monomer contributes to this effect.