Summary A collection of 28 strains of Vibrio cholerae non-O1 isolated during a 3-year period (1989–1991) from hospitalised patients with acute diarrhoea in Calcutta, India, were examined with regard to virulence-associated factors. Of the 28 isolates (each representing a case), 18 were isolated as the sole infecting agent; the remaining 10 were recovered as co-cultures from cases infected with V. cholerae O1. Of the strains isolated in this study, 82% could be serotyped, with serovars O5 (32·1%), O11 and O34 (14·3% each) predominant. Serovars O7, O14, O34, O39 and O97 were associated exclusively with sole infections. Two strains of V. cholerae non-O1 produced anti-cholera toxin IgG-absorbable cholera toxin (CT). Both CT-producing V. cholerae non-O1 strains hybridised with the DNA probe specific for the zonula occludens toxin (ZOT) but none of the remaining 26 strains hybridised with the ZOT probe. The majority of the strains were cytotoxic for CHO, HeLa and Vero cells, with end-point titres of 4–512. Fewer strains produced a cytotonic effect, with end-point titres of 2–16. Of the 28 strains of V. cholerae non-O1 examined, 75%, 75%, 25% and 14·3% produced haemolysin that was active against erythrocytes of rabbit, sheep (Eltor haemolysin), chicken and man, respectively. Strains that produced a haemolysin active against both rabbit and sheep erythrocytes were dominant (35·7%). Ten (35·7%) of the 28 strains examined showed cell-associated haemagglutinating activity on human blood. Of the 10 strains, nine were isolated as sole pathogen and only one strain was associated with mixed infection. Three distinct patterns of inhibition by sugars were detected; inhibition of haemagglutination by mannose 1% but not by fucose and galactose 1% was the dominant haemagglutination inhibition pattern. Six different virulence phenotypes were encountered among strains of V. cholerae non-O1 in this study. The prominent phenotype, which was associated commonly with isolates from patients solely infected by V. cholerae non-O1, was exhibited by strains that produced the Eltor haemolysin, a cytotoxin and a cell-associated haemagglutinin. The production of cell-associated haemagglutinin appeared to be the only distinctive phenotype that could distinguish between isolates from patients solely infected with V. cholerae non-O1 and those associated with mixed infections. From this study, it is apparent that the virulence of V. cholerae non-O1 is multifactorial and mediated by several traits functioning in an integrated fashion. The clinical significance of V. cholerae non-O1 must be assessed in its totality; the presence of a single factor should not be construed as the cause of enteropathogenicity.