We studied the restriction fragment length polymorphism of the rRNA gene and CTX genetic element in Vibrio cholerae O139 Bengal, which resurged in Calcutta in September 1996 after a gap of 32 months. While the strains from this resurgence were indistinguishable from the earlier strains by ribotyping, the structure of the CTX genetic element present in the current O139 strains was found to be unconventional. Vibrio cholerae O139, the second etiological serogroup of cholera, came into being dramatically in September 1992 in Southern India and thereafter spread explosively to all areas of cholera endemicity in India (11) and to neighboring countries (12). Inexplicably, towards the end of 1993 the incidence of V. cholerae O139 declined in Calcutta, India (10), and in other parts of the subcontinent (3), and the O139 serogroup was replaced by a new clone of V. cholerae O1 biotype El Tor (4, 18, 21). However, in September 1996, V. cholerae O139 staged a resurgence. Phenotypically, the reemerged V. cholerae O139 strains were different from those that appeared in late 1992 and 1993 in that the current O139 strains were sensitive to cotrimoxazole (9). However, like the O139 strains isolated in 1992 to 1993 (16), the O139 strains that resurged were resistant to ampicillin, furazolidone, neomycin, and streptomycin (9). Toxigenic strains of V. cholerae contain a compound transposon-like structure known as the CTX genetic element, which comprises a 4.5-kb central core region that contains the ctxAB, zot, ace, orfU, and cep genes and has one or more copies of a 2.7-kb repetitive sequence (designated RS1) flanking it (14, 19). The number and arrangement of the CTX element are known to vary in different strains of toxigenic V. cholerae (20) and have formed a useful basis for the study of the clonality of strains. We embarked on a study to characterize the ribotype, organization, and chromosomal location of the CTX genetic element of the current O139 strains to assess the extent of genotypic variations in these compared to the strains isolated in 1992 to 1993. A total of 10 strains of V. cholerae O139, namely, AS207, AS209, AS210, AS212, AS213, AS231, AS233, AS258, AS259, and AS260, isolated from hospitalized patients with cholera from the Infectious Diseases Hospital, Calcutta, India, between 21 August and 20 September 1996 were selected randomly by blinding the date of isolation from the person who selected the strains. MO1, a serotype O1 strain which could be a progenitor strain of V. cholerae O139 (12), and AP1, an O139 strain which appeared in late 1992, were also included in the study for comparison (13). Chromosomal DNA was prepared (1), restricted with a va