The regular surface protein array (S-layer) present on Aeromonas hydrophila J-1 was composed of a single species of protein of apparent molecular weight 51500. This protein was extracted from whole cells by treatment with 0.2 mol/L glycine hydrocholoide (pH4.0). The protein was purified by Sephadex G-200 gel filtration chromatography and an ion exchange chromatography on DEAE-cellulose. Amino acid composition analysis showed that the protein contained 36.8% hydrophobic amino acids. But the protein did not confer hydrophobicity to the cell surface when present as an intact S-layer by salt aggregation test. ELISA and immunoblotting with two different polyclonal antisera against surface exposed-(PM) and non-surface-exposed epitopes (PR) of the protein revealed that the sensitivity of PM was higher than that of PR. Antigenic diversity of the S-layer proteins from 20 bacterial samples was analysed by ELISA with PM and PF1 (polyclonal antiserum against Aeromonas hydrophila TF7 S-layer protein). The S-layer proteins were distinguishable from the extracellular toxin of the homogeneous strains in antigenic and biochemical characterization and the S-layer proteins were one of the main protective antigens.