In the mismatch repair system of some bacterial and eukaryotic organisms devoid of MutH and Dam methylation, MutL has a divalent cation-dependent endonuclease activity. Recently, our group described that P. aeruginosa MutL protein (PaMutL) has a latent Mn²+ and/or Mg²+ dependent endonuclease activity (Correa et al., 2011). Using supercoiled plasmid, this activity was evidenced by the presence of relax plasmid (single strand break) and to a lesser extent linear DNA (double strand break) as products of the reaction. In order to analyze the requirements of DNA topology/structure for PaMutL endonuclease activity, the reaction was studied using different DNA substrates as linear dsDNA, linear dsDNA containing a single strand loop of different length and circular plasmid with different degree of negative supercoiling. Whereas the hydrolysis of supercoiled plasmid by PaMutL generated nicked and linear DNA products, the treatment of linear dsDNA with PaMutL did not produce internal nicks or double strand breaks. Regarding the degree of supercoiling, PaMutL displayed the same endonuclease activity with all topoisomers analyzed, showing that negative supercoiling did not modified PaMutL endonuclease activity. In addition, while S1 nuclease hydrolyzed all linear dsDNA fragments containing single strand loops, PaMutL was unable to hydrolyze any of them. Our results indicated that PaMutL can hydrolyze closed circular DNA but not linear DNA, suggesting that PaMutL requires fixed DNA molecules ends for its in vitro endonuclease activity.