The unprecedented genesis of a novel non-O1 Vibrio cholerae strain belonging to serogroup O139, which caused an epidemic in late 1992 in the Indian subcontinent, and its subsequent displacement by El Tor O1 vibrios after 18 months initiated a renewed investigation of the aspects of the organism that are related to pathogenesis. The reappearance of V. cholerae O139 with altered antibiotic sensitivity compared to O139 Bengal (O139B) in late 1996 has complicated the epidemiological scenario of V. cholerae and has necessitated an examination of possible rearrangements in the genome underlying such rapid changes in the phenotypic traits. With a view to investigating whether the phenotypic changes that have occurred are associated with alteration in the genome, the genome of the resurgent V. cholerae O139 (O139R) strains were examined. Pulsed-field gel electrophoresis analysis of NotI- and SfiI-digested genomic DNA of O139R isolates showed restriction fragment length polymorphism including in the cholera toxin (CTX) genetic element locus and with O139B isolates. Analyses of the organization of the CTX genetic elements in O139R strains showed that in contrast to two copies of the elements connected by two direct-repeat sequences (RS) in most of the genomes of O139B isolates, the genomes of all O139R strains examined, except strain AS192, have three such elements connected by a single RS. While the RS present in the upstream of the CTX genetic elements in the genome of O139R is of O139B origin, the RS connecting the cores of the elements has several new restriction sites and has lost the BglII site which is supposed to be conserved in all O1 strains and O139B. The endonuclease I-CeuI, which has sites only in the rrn operons in the genomes of all organisms examined so far, has 10 sites in the genomes of O139R strains, compared to 9 in the genomes of O139B strains. The recent isolates of V. cholerae O139 have thus gained one rrn operon. This variation in the number of rrn operons within a serogroup has not been reported for any other organism. The results presented in this report suggest that like the pathogenic El Tor O1 strains, the genomes of O139 strains are undergoing rapid alterations.