The structural gene xds for extracellular DNase of Vibrio cholerae was cloned and inactivated by insertion of the transposon Tn5. The inactivated gene was introduced into the chromosome of V. cholerae by recombination to construct an extracellular DNase-negative strain. Tn5-mediated transposon-facilitated recombination was used to establish the position of xds between the pro-1 and ile-201 markers on the genetic map of V. cholerae. The extracellular DNase-negative strain described here should be useful for investigating the role of the xds-encoded DNase in the physiology of V. cholerae and its plasmids as well as for characterizing other DNases in this organism.