A system for studying gene amplification in the bacterium Acinetobacter sp. strain ADP1 was used to isolate 105 spontaneous mutants. The method selects for the elevated expression of neighboring transcriptional units in a parent strain lacking its normal transcriptional activators. Gene amplification can compensate for the activator loss by increasing the copy number of seven weakly expressed genes. Mutant colonies arose from the parent strain at a frequency of 10(-8) within three weeks. All but one of these mutants carried tandem head-to-tail repeats of a chromosomal segment (amplicon). These amplicons varied in size from approximately 12-290 kb and ranged in copy number from 3 to more than 30. Gene amplification involved a two-step process in which duplications formed independently of recA. Illegitimate recombination fused normally distant chromosomal regions to create novel DNA duplication junctions. These junctions were isolated from amplification mutants using an assay that exploits Acinetobacter natural transformability. Sequence analysis of 72 junctions revealed little identity in the recombining regions. Furthermore, multiple independently isolated mutants contained identical junctions. Six different junctions, each found in two to six mutants, revealed that some recombination events are site-specific. Several recurring junctions were studied using PCR. In each case, the identical duplication present in the mutant was estimated to have occurred in as many as one in a million cells in populations of strains never exposed to selective conditions. These duplications appeared to form spontaneously by a novel type of short homology-independent, site-specific process. However, in the absence of recA, mutant colonies were not selected from parent cells containing these duplications. Thus, the second gene amplification step most likely depends on homologous recombination to increase amplicon copy number. These studies support the theory that gene amplification is a driving force in the evolution of functionally related gene clusters.