Clostridium septicum alpha-toxin is a beta-barrel pore-forming cytolysin that is functionally similar to aerolysin. Residues important in receptor binding, oligomerisation and pore-formation have been identified, however, little is known about the activity of the toxin in an infection, although it is essential for disease. We have now shown that deletion of a small portion of the transmembrane domain, so that the toxin is no longer able to form pores, completely abrogates its ability to contribute to disease, as does substitution of the sole cysteine residue with leucine. However, although previous biochemical and cytotoxicity assays clearly indicated that mutations in residues important in oligomerisation, binding and pre-pore conversion greatly reduced activity or rendered the toxin inactive, once the mutated toxins were over-expressed by the natural host in the context of an infection it was found they were able to confer disease in a mouse model of myonecrosis. These results highlight the importance of testing the activity of virulence determinants in the normal host background and in an infectious disease context, and provide unequivocal evidence that it is the ability of alpha-toxin to form a pore that confers its toxicity in vivo.