The importance of the two major extracellular enzymes of Aeromonas salmonicida, glycerophospholipid: cholesterol acyltransferase (GCAT) and a serine protease (AspA), to the pathology and mortality of salmonid fish with furunculosis had been indicated in toxicity studies. In this study, the genes encoding GCAT (satA) and AspA (aspA) have been cloned and mutagenized by marker replacement of internal deletions, and the constructs have been used for the creation of isogenic satA and aspA mutants of A. salmonicida. A pSUP202 derivative (pSUP202sac) carrying the sacRB genes was constructed to facilitate the selection of mutants. The requirement of serine protease for processing of pro-GCAT was demonstrated. Processing involved the removal of a short internal fragment. Surprisingly, pathogenicity trials revealed no major decrease in virulence of the A. salmonicida delta satA::kan or A. salmonicida delta aspA::kan mutants compared to the wild-type parent strains when Atlantic salmon (Salmo salar L.) were challenged by intraperitoneal injection. Moreover, using a cohabitation model, which more closely mimics the natural disease, there was also no significant decrease in the relative cumulative mortality following infection with either of the deletion mutants compared to the parent strain. Thus, although these two toxins may confer some competitive advantage to A. salmonicida, neither toxin is essential for the very high virulence of A. salmonicida in Atlantic salmon. This first report of defined deletion mutations within any proposed extracellular virulence factor of A. salmonicida raises crucial questions about the pathogenesis of this important fish pathogen.