We have examined the possibile relationships between extracellular protease and haemolysin and virulence in Aeromonas salmonicida, and the possible plasmid-encoded origin of these extracellular enzyme activities. Variants, isolated from different strains, showed simultaneous loss of both protease and haemolysin. The frequency of spontaneous loss from one strain (0.1–0.4%) as well as the frequency of induced loss from four strains (0.3–18%) treated with ethidium bromide suggested a plasmid-encoded origin for these enzymes. However, plasmid analyses showed no detectable loss of plasmids correlated with the loss of the extracellular activities. There was no change in the LD100 when fish were exposed to one virulent strain or its protease, haemolysin-negative variants indicating that these extracellular activities are not necessary for virulence or pathogenicity in the acute form of furunculosis. Furthermore, both protease, haemolysin-negative and protease, haemolysin-positive clones isolated from a second virulent strain treated with ethidium bromide were avirulent (LD50 increased >4 logs). Thus, attenuation of A. salmonicida can occur without detected loss of the extracellular enzymes, plasmids, or the A-layer.