After transfection of amplification-promoting DNA elements into mammalian cells, homogeneously staining regions (HSRs) are formed by high copy numbers of transfected DNA arranged in head-to-tail polymers. Here, we wanted to evaluate the stability of this type of HSR during prolonged cultivation of transfected cells in selective medium. Thymidine kinase-deficient mouse L cells were transfected with pAPR4tk DNA harboring the amplification-promoting element 4 (APR4) linked to the gene for thymidine kinase (TK) or, alternatively, transfected with a DNA construct (pARP4t-PA) carrying, in addition, the expression cassette for human tissue-type plasminogen activator (t-PA). After transfection, one or two HSRs per cell were formed that disintegrated spontaneously after 25-40 wk of continuous cultivation in the presence of selective HAT (hypoxanthine-aminopterin-thymidine) medium. Unexpectedly, plasmid DNA reinserted into a plethora of new chromosomal sites, as revealed by in situ hybridization and Southern blot analysis. Coincidently, secretion of t-PA decreased to 10-20% of its original level. After transfection of pAPR4tk DNA lacking the t-PA expression cassette, HSR decay and reintegration of plasmid constructs into multiple chromosomal sites were also observed, whereas the ptk vector without an amplification-promoting DNA element did not form an HSR after transfection. We conclude that, in contrast to the pattern of known structures with head-to-tail arrangements, the HSR formed by amplification-promoting DNA elements represents a novel type of HSR that disintegrates by transposition into a plethora of new chromosomal integration sites. This process is mediated by the amplification-promoting DNA element itself and can be observed even when selective pressure is maintained.