The predominant cell surface protein (A protein) of Aeromonas salmonicida has been purified in high yield from outer membranes by using a combination of detergent and chaotropic extraction methods as well as exclusion and ion-exchange chromatography. The A protein was primarily monomeric, Mr 50 000, but readily formed oligomers at high protein or low salt concentrations. Several isoelectric forms were distinguishable with purified protein as well as in situ on the cell surface. Neither phosphate nor carbohydrate was detectable. The A protein was hydrophobic in composition and the N-terminal sequence highly hydrophobic. From CD spectra the A protein exhibited 14% alpha helix and 19-28% beta structure and could also be readily crystallized. By fluorescent antibody staining the A protein was shown to cover the entire cell surface but was absent from A protein deficient mutants. This protein appears to have no apparent enzymatic activity but rather constitutes a macromolecular refractile protein barrier essential for virulence.