Of 167 Vibrio cholerae isolates screened for sequences homologous with zonula occludens toxin (zot) or cholera toxin (ctx) genes, 3.0% of non-O1, 100.0% of clinical O1, and 0.0% of environmental O1 strains contained both zot and ctx. zot was present only in strains that were ctx positive; all ctx-positive strains carried zot. The absence of zot-positive, ctx-negative strains suggests ZOT is not an independent virulence factor for V. cholerae, although ZOT may play a role in the pathogenesis of toxigenic strains.