Until recently, only Vibrio cholerae strains of the O1 serogroup have been associated with epidemic cholera. In December 1992, an outbreak of cholera gravis in Vellore, India, was attributed to a new serogroup of V. cholerae recently designated O139. Serogroup O139 cholera has since spread to 13 countries and has reached pandemic proportions. Serogroup O139 cholera evades immunity to O1 cholera and is not detected by the standard O1 antigen test. Understanding the origins of O139 cholera and determining the relatedness of O139 to O1 cholera are necessary to device strategies for detecting, reporting, and controlling this new pandemic. In order to determine the origins of this novel cholera serogroup, O139 was analyzed for virulence genes, for virulence proteins and their regulation, and for its genomic background. We found that O139 and O1 V. cholera strains of the E1 Tor biotype possess highly homologous virulence genes encoding cholera toxin and toxin-coregulated pili and that the regulation of virulence protein expression likewise was indistinguishable between O139 and O1. Pulsed-field gel electrophoresis (PFGE) revealed the restriction digest pattern of O139 strains to be closely related to that of O1 serogroup E1 Tor biotype cholera strains from the Indian subcontinent. However, PFGE showed minor differences among individual O139 cholera isolates, suggesting that O139 V. cholerae is evolving.