The organization of amplified DNA in mammalian cells in the form of inverted repeats rather than tandem repeats was first observed and studied in the 3B rat cell line. The structure and chromosomal location of the amplified inverted duplications in this cell line have been further analyzed by cloning, long-range mapping, and fluorescence in situ hybridization. The amplification unit is at least 450 kilobases in size and all of the amplicons are located in a single chromosomal location of approximately 10 or 11 megabases. No heterogeneity in either size or molecular structure is detected between the 3B amplicons, indicating that the 20- to 40-fold amplification occurred in a single event and not through a series of events, which would result in heterogeneity among the amplicons. Thus the amplification in 3B cells may reflect more closely the situation seen in tumors containing amplified oncogenes/protooncogenes than the amplifications present in cell lines after multiple selections with cytotoxic drugs. The progenitor Rat-2 cell line contains three alleles of the region of DNA that is amplified in 3B cells; two are located on the two normal homologues of rat chromosome 2 and the third is at the equivalent position on a marker chromosome, der(3)t(2;3). 3B cells contain only one of the two normal homologues of chromosome 2 in addition to chromosome der(3)t(2;3). All of the amplified DNA is located on a new marker chromosome, M2, whose amplified DNA region does not resemble chromosome 2. These results are consistent with the amplification model proposed by Passananti et al. [Passananti, C., Davies, B., Ford, M. & Fried, M. (1987) EMBO J. 6, 1697-1703], in which the excision from a chromosome of the DNA to be amplified results in the loss of rearrangement of that chromosome. In this model the excised DNA can be amplified extrachromosomally during a single S phase before becoming stabilized by integration into a chromosome, probably at a different location to that of its unamplified allele.