Enteropathogenic Escherichia coli (EPEC) utilises a type III secretion system to translocate effector proteins directly into enterocytes and subvert cell signaling. Non-LEE encoded effector C (NleC) is one of a number of effectors which inhibit NF-κB signaling. The mechanism of action of NleC is unlike those of effectors such as NleB or NleE which inhibit NF-κB signaling upstream of NF-κB proteins. NleC acts directly on the NF-κB protein p65 by cleaving it and preventing inflammatory cytokine production to allow EPEC to persist within the host. Analysis of the amino acid sequence of NleC reveals it contains the zinc metalloprotease consensus motif HEXXH. Cleavage of p65 has been shown to occur within the Rel homology domain (RHD) of p65 which is highly conserved among all NF-κB proteins. Here we show that NleC cleaves another Rel protein, p50, within the RHD. This study also aims to identify the regions of binding between p65 and NleC. We show that NleC is likely to have two binding sites within p65 (between amino acids 22-26 and 170-184). We determined the minimum region of p65 bound and cleaved by NleC to be the DNA-binding sub-domain of p65, which is 180 residues long. Smaller peptides of p65 are unable to be cleaved by NleC suggesting that the protease recognizes a larger, folded protein subunit. Current studies are underway to further define this binding and obtain a better understanding of substrate recognition by NleC.