Organophosphate degrading enzymes are of great interest in light of their ability to detoxify chemical warfare agents. The diisopropylfluorophosphatase (DFPase) from Loligo vulgaris is characterized by its high stability and broad substrate specifity. Here we report the production of large amounts of active, recombinant DFPase using an Escherichia coli expression system. The enzyme was purified to homogeneity using a combination of immobilized metal affinity and ion exchange chromatography. CD-spectroscopy indicates a well folded protein with a high amount of beta -sheet structure. Limited proteolysis was used to gain a deeper insight into the structural organization of the protein. DFPase possesses an internal protease-sensitive region located between amino acids 146 and 149. The two proteolytic fragments remain as a tight complex retaining a DFPase activity comparable to the intact enzyme. Overexpression clones for each fragment were constructed with the expression resulting in the formation of inclusion bodies. Upon isolation and refolding active protein is only formed when both fragments are present. Thus, the two proteolytic fragments are probably part of a stable single-domain protein with multiple contacts between them and only one protease accessible surface loop.