Abstract: Methods of isolating deletion mutants of Vibrio cholerae. In one method, the deletion is predetermined by digestion with restriction endonucleases of known specificity. The deletions are inserted into the Vibrio cholerae chromosome by in vivo recombination between a plasmid carrying the desired deletion, with adjacent flanking sequences, and the Vibrio cholerae chromosome. In another method, an initial in vivo recombination event of homologous sequences from the recombinant plasmid into the chromosome provides a selectable marker at this site. A second in vivo recombination event between homologous flanking sequences results in excision of proficient genes from the chromosome with the end product being a deletion mutation. Also provided are methods for the isolation and characterization of a new Vibrio cholerae strain having a deletion in the ctx gene, as defined by Acc I, Xba I, Cla I and/or restriction endonuclease sites and further having a deletion in the gene encoding zonula occludens toxin (zot).