The recent spread of El Tor cholera to America augments the need for an effective, safe and economical vaccine. In the present paper we describe the construction of live attenuated V. Cholerae strains by specifically deleting the genes encoding cholera toxin and other putative toxins from the bacterial chromosome. To maximize the likelihood of exposing protective antigens relevant to currently circulating vibrios we selected for genetic manipulation recent epidemic V. cholerae isolates from Peru. The mutant strains did not produce cholera toxin in vitro and in vivo. Deletion of the virulence cassette was accompanied by marked attenuation in the infant mouse cholera model. A selected El Tor Ogawa candidate vaccine strain was refractory to acquisition of foreign genes by conjugation with toxigenic vibrios.