The recently described type III secretion system (TTSS) of Aeromonas salmonicida subsp. salmonicida has been linked to virulence in salmonids. In this study, three TTSS effector genes, aexT, aopH or aopO, were inactivated by deletion, as was ascC, the gene encoding the outer-membrane pore of the secretion apparatus. Effects on virulence were assayed by live challenge of Atlantic salmon (Salmo salar). The ascC mutant strain was avirulent by both intraperitoneal (i.p.) injection and immersion, did not appear to establish a clinically inapparent infection and did not confer protection from subsequent rechallenge with the parental strain. 1H NMR spectroscopy-based metabolite profiling of plasma from all fish showed significant differences in the metabolite profiles between the animals exposed to the parental strain or ascC. The experimental infection by immersion with aopO was indistinguishable from that of the parental strain, that of aexT was delayed, whilst the virulence of aopH was reduced significantly but not abolished. By i.p. injection, aexT, aopH and aopO caused an experimental disease indistinguishable from that of the parental strain. These data demonstrate that while the TTSS is absolutely essential for virulence of A. salmonicida subsp. salmonicida in Atlantic salmon, removal of individual effectors has little influence on virulence but has a significant effect on colonization. The ascC i.p. injection data also suggest that in addition to host invasion there is a second step in A. salmonicida pathogenesis that requires an active TTSS.