Clostridium septicum is the causative agent of spontaneous gas gangrene or atraumatic myonecrosis, a sudden and frequently fatal infection that is increasingly associated with malignancy of the colon. Little is known about the disease process although the focus of virulence studies has been the alpha-toxin, a pore-forming cytolysin that is encoded by the csa gene and secreted as an inactive protoxin. Until now a lack of techniques for the genetic manipulation of C. septicum has hindered the use of molecular approaches to understand pathogenesis. By introducing plasmids by conjugation from Escherichia coli, we have developed methods for the genetic manipulation of C. septicum and constructed a chromosomal csa mutant by allelic exchange. Virulence testing of an isogenic series of strains consisting of the wild type, the csa mutant, and a csa mutant complemented with the wild-type csa gene revealed that the development of fulminant myonecrosis in mice was dependent on the ability to produce a functional haemolytic alpha-toxin. Furthermore, the inhibition of leukocyte influx into the lesion, which is very typical of clostridial myonecrosis, was also dependent on the ability to produce alpha-toxin. This study represents the first definitive identification of a virulence factor in this organism and opens the way for further studies that will delineate the role of other putative virulence factors in this significant pathogen.