Toxigenic Vibrio cholerae strains are lysogens of CTXPhi, a filamentous phage which encodes cholera toxin. The receptor for CTXPhi for invading V. cholerae cells is the toxin-coregulated pilus (TCP), the genes for which reside in a larger genetic element, the TCP pathogenicity island. We analyzed 146 CTX-negative strains of V. cholerae O1 or non-O1 isolated from patients or surface waters in five different countries for the presence of the TCP pathogenicity island, the regulatory gene toxR, and the CTXPhi attachment sequence attRS, as well as for susceptibility of the strains to CTXPhi, to investigate the molecular basis for the emergence of new clones of toxigenic V. cholerae. DNA probe or PCR assays for tcpA, tcpI, acfB, toxR, and attRS revealed that 6.85% of the strains, all of which belonged to the O1 serogroup, carried the TCP pathogenicity island, toxR, and multiple copies of attRS, whereas the remaining 93.15% of the strains were negative for TCP but positive for either one or both or neither of toxR and attRS. An analysis of the strains for susceptibility to CTXPhi, using a genetically marked derivative of the phage CTX-KmPhi, showed that all TCP-positive CTX-negative strains and 1 of 136 TCP-negative strains were infected by the phage either in vitro or in the intestines of infant mice. The phage genome integrated into the chromosome of infected V. cholerae O1 cells forming stable lysogens. Comparative analysis of rRNA gene restriction patterns revealed that the lysogens derived from nontoxigenic progenitors were either closely related to or distinctly different from previously described clones of toxigenic V. cholerae. To our knowledge, this is the first demonstration of lysogenic conversion of naturally occurring nontoxigenic V. cholerae strains by CTXPhi. The results of this study further indicated that strains belonging to the O1 serogroup of V. cholerae are more likely to possess the TCP pathogenicity island and hence to be infected by CTXPhi, leading to the origination of potential new epidemic clones.