Essentially all eukaryotic cells contain circular extrachromosomal DNA as a result of excision from the chromosomes. To obtain insight into the nature of recombination associated with the occurrence of such DNA species and its biological significance, we analyzed a library enriched in recombination junctions which was constructed by a novel DNA subtraction technique; in-gel competitive reassociation (IGCR). Furthermore, we also introduced inverse PCR to characterize chromosomal DNA fragments containing the recombination junctions. At least 45% of the clones in the library constructed by the IGCR procedure comprised DNA with recombination junctions. Nucleotide sequence analysis of the recombination junctions indicated that three of four extrachromosomal DNAs thus analyzed were produced through recombination between sequences with a 3-5 bp homology in the chromosomes. One extrachromosomal DNA was apparently generated through non-homologous recombination, possibly by end-to-end joining. These results have demonstrated the usefulness of IGCR in concentrating recombination junctions, which provide the most direct evidence for the mechanism of the recombinational events involved, from highly complex genomes.