Biochemical characteristics, SDS-PAGE analysis of whole cell proteins and maximum growth temperature were used for phenotypic characterization of the three DNA hybridization groups (HG 1, HG 2 and HG 3) of the Aeromonas hydrophila complex (96 strains). DNA-DNA hybridization was made with 22 strains using both P-32- and digoxigenin-labelled probes. The strains originated from raw foods of animal origin, drinking water, fresh water, diseased fish and clinical material. The biochemical tests which were shown to be of value in the separation of the three hybridization groups (HG) were DL-lactate and urocanic acid utilization and acid production from D-sorbitol and from D-rhamnose. A new marker, the maximum growth temperature determined with a temperature-gradient incubator, was shown to separate HG 1 from HG 2 and HG 3. The mean maximum growth temperature (T,,,) of confirmed and putative HG 1 strains was significantly higher (41.1 +/- 0.56 degrees C; P < 0.001) than that of HG 2 (38.2 +/- 0.79 degrees C) or HG 3 (38.6 +/- 0.75 degrees C) strains. The lower T-max of HG 2 and HG 3 may explain why the results of biochemical tests may differ if incubation temperatures of 30 degrees or 37 degrees C are used. The SDS-PAGE protein pattern analysis indicated that the strains of HG 3 have a band of variable molecular weight of 24, 25 or 26 kDa not possessed by strains of HG 1 and HG 2. HG 2 strains had two weak bands of molecular weight 24 and 25 kDa. HG 1 organisms were isolated from ground beef, chicken and clinical material but not from fresh water or drinking water. HG 2 and HG 3 organisms were isolated from foods of animal origin and from the water samples.