In this study, the phenotypic, antigenic, and virulence characteristics of 32 Citrobacter freundii strains of fish, human, and veterinary origin were comparatively analyzed. In addition, the spread of drug resistance factors by conjugation was investigated. Regardless of the source of isolation, the strains exhibited variable reactions mainly for arginine dihydrolase, ornithine decarboxylase, and fermentation of sucrose, melibiose, amygdalin, and salicin. Total fatty acid methyl ester analysis by gas chromatography proved to be useful for an intratypic differentiation within the C. freundii strains studied. In fact, although all of the isolates exhibited similar fatty acid methyl ester profiles, significant differences in the major fatty acids 16:1 and 16:0 and in the 17:0 delta region were observed between the isolates from salmonids and the remaining strains. Serological studies using agglutination tests, analysis of lipopolysaccharides (LPS), and the corresponding immunoblots with 13 antisera indicated a great antigenic diversity among the strains. Common LPS patterns were shared only by some isolates showing high cross-agglutination titers. In contrast, although all strains exhibited very similar surface protein patterns, only two common outer membrane proteins of 54 and 58 kDa were immunologically related. Infectivity trials performed in mice and rainbow trout indicated that all of the C. freundii strains were not pathogenic for mice (50% lethal dose of > 5 x 10(7)). Although the isolates displayed a low degree of virulence for trout, inoculated strains were always recovered from the survivors in pure culture.(ABSTRACT TRUNCATED AT 250 WORDS)