The flap endonucleases are involved in a wide range of processes involving the structure-specific cleavage of branched DNA and RNA substrates. They are metalloenzymes, able to hydrolyse DNA and RNA substrates from the 5’-end liberating mono-, di- and polynucleotides with a 5’-phosphate. Much effort has focussed on analysis of the contribution of a number of highly conserved residues involved in metal binding, substrate interactions and catalysis (Feng et al). A group of cryptic flap endonuclease paralogues can be identified using homology searches. It has been reported that the flap endonuclease paralogue present in E. coli known as Exo IX is able to digest DNA in the 3’-5’ direction, i.e. opposite to the usual polarity of flap endonuclease activity. We wish to present the results of our study on this unusual protein and an orthologue found in the human pathogen Staphylococcus aureus. The E. coli Exo IX protein was shown to possess a weak 3’-5’ exonucleolytic activity (Shafritz et al). It was able to interact with a range of proteins known to bind to single-stranded and branched DNA. These proteins were identified using a combination of affinity chromatography on Exo IX-Spepharose beads with tryptic digest MALDI-TOF. The S. aureus enzyme was found to behave as a classical flap endonuclease and was able to hydrolyse a wide range of structured DNA substrates. We will reveal the mechanism by which Exo IX is apparently able to hydrolysis DNA in reverse!