Environmental isolates of Vibrio cholerae of eight randomly amplified polymorphic DNA (RAPD) fingerprint types from Calcutta, India, that were unusual in containing toxin-coregulated pilus or cholera toxin genes but not O1 or O139 antigens of epidemic strains were studied by PCR and sequencing to gain insights into V. cholerae evolution. We found that each isolate contained a variant form of the VPI pathogenicity island. Distinguishing features included (i) four new alleles of tcpF (which encodes secreted virulence protein; its exact function is unknown), 20 to 70% divergent (at the protein level) from each other and canonical tcpF; (ii) a new allele of toxT (virulence regulatory gene), 36% divergent (at the protein level) in its 5 half and nearly identical in its 3 half to canonical toxT; (iii) a new tcpA (pilin) gene; and (iv) four variant forms of a regulatory sequence upstream of toxT. Also found were transpositions of an IS903-related element and function-unknown genes to sites in VPI. Cholera toxin (ctx) genes were found in isolates of two RAPD types, in each case embedded in CTX-like prophages. Fragments that are inferred to contain only putative repressor, replication, and integration genes were present in two other RAPD types. New possible prophage repressor and replication genes were also identified. Our results show marked genetic diversity in the virulence-associated gene clusters found in some nonepidemic V. cholerae strains, suggest that some of these genes contribute to fitness in nature, and emphasize the potential importance of interstrain gene exchange in the evolution of this species.Vibrio cholerae is a genetically diverse species that lives in warm-water environments, often associated with plankton and other aquatic organisms (9, 15, 31). Strains of just two of the approximately 200 currently known O-antigen serogroups (50; T. Shimada, personal communication), O1 and O139, cause epidemic cholera-the acute, devastating diarrheal disease that afflicts many thousands of people annually, especially in developing countries. Although certain other serogroups cause sporadic diarrheal disease (30, 37), most V. cholerae organisms probably do not infect humans. Epidemic strains are also distinguished from most others by their production of cholera toxin and a toxin-coregulated pilus (TCP) (15,21).Epidemic strains of V. cholerae have a complex natural history in areas of cholera endemicity, involving rapid transmission to humans via contaminated food and water during each year's cholera season and persistence or proliferation in aquatic organisms or abiotic niches at other times. Several recent major changes in patterns of epidemic cholera may stem in large part from a combination of human factors and environmental fluctuation that could affect the distribution of aquatic host species. The bacterial changes include (i) the replacement of classical biotype O1 strains beginning in 1961 by strains of the new El Tor O1 biotype (15), (ii) the reemergence of classical biotype strains in parts of Bangladesh i...