The asoB gene of Aeromonas salmonicida is located approximately 9 kb downstream of the structural gene (vapA) for the surface layer (A-layer). The nucleotide sequence of asoB was determined and found to encode a putative polytopic cytoplasmic membrane protein which exhibited homology to a number of bacterial transport proteins. Allele exchange mutagenesis of asoB resulted in a mutant (A449-D) which was avirulent when administered by bath immersion. However, when administered by intraperitoneal injection, A449-D is as lethal as wild type. Characterization of the phenotype of A449-D showed that there were pleiotropic effects on VapA secretion, haemolysis and outer membrane protein composition. Mobilization of cloned asoB on a broad-host-range plasmid into A449-D resulted in the complementation of VapA translocation, haemolytic activity and virulence.