Clostridium perfringens is an anaerobic bacterium that causes avian necrotic enteritis, which is characterised by damage to the intestinal mucosa. We previously showed that the secreted pore-forming toxin, NetB, is a major virulence factor in the pathogenesis of necrotic enteritis in chickens. The netB gene is encoded on a conjugative plasmid that very unusually co-exists in the same cell as two other very closely related conjugative plasmids, all of which have essentially the same conjugation locus. NetB has amino acid sequence similarity to a-hemolysin, a b-barrel pore-forming toxin from Staphylococcus aureus. These toxins are produced as water soluble monomers that upon target cell recognition oligomerise and undergo conformational changes to produce a transmembrane pore that causes cell lysis. We have determined the crystal structure of the water soluble form of NetB to 1.8 Å. The results showed that it adopts a similar fold to a-hemolysin, however, there are key differences in the conformation of the membrane binding domain, indicating that NetB may recognise different membrane receptors or use a different mechanism for membrane-protein interactions. Using the ability of NetB to lyse red blood cells as a screen, we have carried out a random mutagenesis study that identified several residues that are critical for NetB-induced cell lysis. Key residues identified by the mutagenesis studies were mapped onto the crystal structure; most of these amino acids clustered in regions predicted to be required for oligomerisation or membrane binding. These data provide an insight into the mechanism of NetB pore formation and will contribute to our understanding of the mode of action of this important toxin.