Insertion sequences (ISs) are the smallest autonomous mobile genetic elements being typically <2.5kb long, with a single open reading frame encoding a transposase bounded by very short inverted repeats. Despite their simplicity ISs profoundly influence bacterial genomes by promoting intragenomic rearrangements that can lead to clustering of genes and formation of more complex mobile elements. This is especially relevant to the evolution of multidrug resistance. We explore the IS1111-attC subgroup as a model for this process. The IS1111-attC subgroup of ISs are believed to specifically target the attC recombination sites of integrons and are strongly associated with members of the genus Pseudomonas harboring chromosomal integrons. IS1111-attC has recently invaded recombination sites of mobile class 1 integrons in genera other than Pseudomonas. Conditions affecting the rate of IS1111-attC movement between environmental and clinical Pseudomonads are not known. Transposition of IS1111-attC has been directly detected in E. coli but not Pseudomonas. The inferred mechanism for IS1111-attC transposition is site-specific recombination. The significance of this transposition has been difficult to examine due to the lack of an assay assessing IS1111-attC activity. We used natural transformation experiments in Ps. stutzeri ATCC17587, and electrophoretic mobility shift assays (EMSAs) using purified transposase, to study activity of IS1111-attC in vivo and in vitro. We captured an IS1111-attC transposition by transforming a synthetic ‘attC-trap’ plasmid into a Ps. stutzeri strain harbouring IS1111-attC elements. In the absence of selective pressure, transposition was detected in 2% of Ps. stutzeri attC-trap transformants. Our EMSA data suggests that, unlike the integron integrase, the IS1111-attC transposase preferentially binds the 5’ attC strand from the native ATCC17587 strain. Like the integron integrase, this transposase does not bind to double stranded attC DNA. This is the first evidence for IS1111-attC activity in Pseudomonas cells and illustrates the potential for these elements to move between chromosomal and plasmid borne integrons.