Infection of human cells with adenovirus serotype 12 (Ad12) induces metaphase fragility of four, and apparently only four, chromosomal loci. Surprisingly, each of these four loci corresponds to a cluster of genes encoding a small abundant structural RNA: the RNU1 and RNU2 loci contain tandemly repeated genes encoding U1 and U2 small nuclear RNAs (snRNAs), respectively; the PSU1 locus is a cluster of degenerate U1 genes; and the RN5S locus contains the tandemly repeated genes encoding 5S rRNA. These observations suggested that high local levels of transcription, in combination with Ad12 early functions, can interfere with metaphase chromatin packing. In support of this hypothesis, we and others found that an artificial tandem array of transcriptionally active, but not inactive, U2 snRNA genes would generate a novel Ad12-inducible fragile site. Although U1 and U2 snRNA are both transcribed by RNA polymerase II and share similar enhancer, promoter, and terminator signals, the human U1 promoter is clearly more complex than that of U2. In addition, the natural U1 tandem repeat unit exceeds 45 kb, whereas the U2 tandem repeat unit is only 6.1 kb. We therefore asked whether an artificial array of minimal U1 genes would also generate a novel Ad12-inducible fragile site. The exogenous U1 genes were marked by an innocuous U72C point mutation within the U1 coding region so that steady-state levels of U1 snRNA derived from the artificial array could be quantified by a simple primer extension assay. We found that the minimal U1 genes were efficiently expressed and were as effective as minimal U2 genes in generating a novel Ad12-inducible fragile site. Thus, despite significant differences in promoter architecture and overall gene organization, the active U1 transcription units suffice to generate a new virally inducible fragile site.