Transposition events mediated by plasmidborne copies of the insertion sequence IS3 of Escherichia coli are difficult to detect because of a low frequency of cointegrate formation. We found that cointegration activity could be strongly enhanced by using plasmid constructions in which a second IS3 element, disabled by a large deletion, was placed adjacent to an intact IS3 copy. Attempts to construct plasmids containing two adjacent intact IS3 copies were unsuccessful, probably because of instability. Transpositional hyperactivity of tandemly duplicated IS sequences was previously described for spontaneous duplications of IS21 and IS30 and may well be a more general phenomenon. The frequency of cointegration events was also strongly increased in an E. coli strain deficient in Dam methylation, suggesting that IS3, like some other Dam site-containing IS elements, is regulated by the Dam methylation system. Insertion sites were strongly clustered within the target lambda repressor gene: however no sequence specificity determinants could be identified. All insertions analyzed carried the IS element in the same orientation; target sequence duplications were mostly 3 bp, but in some cases 4 bp long. To obtain information about the roles of the open reading frames (ORFs) in IS3, we constructed plasmid-borne mutant elements in which potentially functional reading frames were inactivated by site-directed mutations; the mutants were introduced into partial tandem constructions and tested in cointegration assays. Mutations inactivating the putative initiation condons of ORF I and II in the intact element reduced insertion activity to less than 4% of the wild type, whereas the introduction of a termination codon into ORF IV had no effect on cointegration frequency.(ABSTRACT TRUNCATED AT 250 WORDS)