The El Tor biotype of Vibrio cholerae has several characteristics that differentiate it from the classical biotype of V. cholerae. Among these are production of soluble hemolysin(s), a cell-associated hemagglutinin for chicken erythrocytes, resistance to polymyxin B, and resistance to bacteriophages of Mukerjee group IV. In the present study, we located the determinants for hemolysin (hly), chicken erythrocyte hemagglutinin (cha), and polymyxin B resistance (pmx) on the genetic map of V. cholerae. Transposon-facilitated recombination was used to perform conjugal matings between El Tor donor strains and classical recipient strains of V. cholerae. Recombinants were selected that had inherited specific nutritional markers from the donor strains and streptomycin resistance from the recipient strains. The recombinants were tested for the presence or absence of the unselected donor markers hly, cha, and pmx. These three El Tor biotype markers were found to be closely linked to each other and were located between the pyrA-201 and his-2 loci on the genetic map of V. cholerae.