The insertion sequence IS21 (2.1 kb) originating from the broad-host-range IncP plasmid R68 transposes infrequently; by contrast, the IS21 tandem repeat found on the derivative R68.45 is highly active in transpositional mobilization of other replicons in a variety of Gram-negative bacteria. The mobilized plasmids are joined to R68.45 by single IS21 copies in direct orientation. The formation of IS21 tandem duplications was observed in cointegrates between R68.45 and pBR325::IS21 and also in an RP1::IS21 plasmid derivative in which a segment located between two directly repeated copies of IS21 was deleted spontaneously. We speculate that IS21 tandem repeats can arise when the termini of two IS21 elements are specifically joined in a transposition or deletion event. A resistance gene flanked by two IS21 elements in direct orientation did not behave as a transposon. The omega fragment carrying transcription and translation stop signals was inserted into various sites of the IS21 tandem repeat; in this way it could be shown that the left IS21 element (which is next to the kanamycin resistance gene in R68.45) was 100 times more active in cointegrate formation than was the right-hand element. Cointegrates between the conjugative plasmid R751 and pBR325 derivatives carrying IS21 and IS21::omega in tandem contained a single IS21 at one replicon junction and a single IS21::omega at the other. In the IS21 duplications the inner IS21 ends were preferentially recognized (presumably by IS21 transposase), whereas the outer termini were not required for cointegrate formation. Based on these findings a conservative (simple) pathway of transposition is proposed for R68.45 and other plasmids with an IS21 tandem repeat.(ABSTRACT TRUNCATED AT 250 WORDS)