Amplification of sequences within mammalian chromosomes is often accompanied by the formation of homogeneously staining regions (HSRs). The arrangement of DNA sequences within such amplicons has been investigated, but little is known about the chromosome structure or behaviour of these unusual regions. We have analysed the metaphase chromosome structure of the dihydrofolate reductase (DHFR) amplicon of CHOC400 cells. The chromatin in this region contains hyperacetylated nucleosomes yet, at the same time, appears to be densely packed like heterochromatin. The region does not bind heterochromatin proteins. We show that the dense packing of the region is restricted to DNA located close to the chromosome core/scaffold. In contrast, levels of the chromosome scaffold protein topoisomerase II at HSRs are the same as those found at other euchromatic locations. Metaphase chromosome condensation of the HSR is shown to be sensitive to topoisomerase II inhibitors, and sister chromatids often appear to remain attached within the HSRs at metaphase. We suggest that these features underlie anaphase bridging and the aberrant interphase structure of the HSR. The DHFR amplicon is widely used as a model system to study mammalian DNA replication. We conclude that the higher-order chromosome structure of this amplicon is unusual and suggest that caution needs to be exercised in extrapolating data from HSRs to normal chromosomal loci.