IS492 transposition in the marine bacterium Pseudoalteromonas atlantica controls the phase variation of extracellular polysaccharide (EPS). IS492 inserts into a single site in epsG, a predicted glycosyl transferase gene important for EPS production, and precisely excises to form a circular element. This reversible insertion of IS492 into epsG is mediated by the DEDD recombinase MooV. Recombinases in the novel Piv/MooV family perform site-specific recombination and DNA transposition, an unprecedented characteristic for a single recombinase family, however the recombination mechanism used by these recombinases remains to be elucidated. The goal of this research was to characterize the recombination mechanism used in MooV-mediated IS492 transposition. This study demonstrates that MooV-mediated IS492[Delta]mooV::tet insertion in E. coli is facilitated by using temperatures close to the growth optimum for P. atlantica. Single IS492[Delta]mooV::tet insertion into the epsG site duplicates the 5-bp 5-CTTGT-3 sequence. Tandem insertion into the IS492[Delta]mooV::tet donor plasmid also targets this 5-bp sequence flanking the element, where the tandem element, (IS492[Delta]mooV::tet)2, is separated and flanked by 5-CTTGT-3. This suggests that the mechanism for insertion is either site-specific recombination or cut-and-paste transposition where the 5-bp sequence is the common core of the recombination sites or the recognition sequence for the staggered cut by MooV, respectively. Chromosomal insertions were not observed, suggesting a target site preference. Experiments using the (IS492[Delta]mooV::tet)2 with the epsG target plasmid show that MooV-mediated transposition of (IS492[Delta]mooV::tet)2 is less dependent on lower temperatures and is more active for transposition compared to IS492[Delta]mooV::tet. Precise excision of one IS492[Delta]mooV::tet from the donor plasmid was observed, and the most frequent insertion products were cointegrates that had two directly-oriented IS492[Delta]mooV::tet elements flanking the target and donor plasmids with 5-CTTGT-3 at the junctions. These results suggest that the inner ends of (IS492[Delta]mooV::tet)2 formed the active site for targeted insertion into the epsG site by either a site-specific or cut-and-paste mechanism, and that the rate-limiting step in IS492 transposition is the formation of the circle-junction sequence.