Sixty-four representative strains of Vibrio cholerae O139 were analyzed, to re-examine the origin of this serogroup. Ribotyping differentiated the strains into 3 HindIII and 7 BglI ribotypes. One HindIII and 5 BglI ribotypes were shared by all toxigenic O139 strains. Of 6 nontoxigenic O139 strains, 3 shared ribotypes with the toxigenic strains, carried genes encoding toxin coregulated pilus, and were susceptible to the cholera toxin-converting bacteriophage CTXPhi. The remaining 3 strains belonged to 2 different ribotypes distinct from toxigenic O139 strains and were resistant to CTXPhi and JA-1, an O139-specific lytic bacteriophage. Polymerase chain reaction amplicons corresponding to the gmhD gene carried by these 3 strains also differed from those of the toxigenic O139 strains but were identical to those of 15 environmental non-O1-non-O139 strains. Thus, the O139 antigen is present in different lineages, and this serogroup appears to comprise epidemic and nonepidemic strains derived separately from different progenitors.