In Vibrio cholerae, the genes encoding cholera toxin (ctxAB) are located on a segment of DNA (termed the "core" region) that is flanked by two or more copies of a repeated sequence called RS1. Together these DNA units comprise the CTX genetic element. Evidence presented here suggests that RS1 sequences encode a site-specific recombination system, which allows integration of a suicide plasmid carrying RS1 into an 18-base-pair sequence (attRS1) located on the chromosome of nontoxigenic V. cholerae strains. Strains of V. cholerae with large deletions removing attRS1 and the entire CTX genetic element no longer undergo site-specific recombination with the RS1 sequence. Additionally, these deletion strains show a defect in intestinal colonization. Recombination experiments localize the gene responsible for enhancing colonization to a portion of the core region of the CTX element. The identified gene encodes a peptide that is highly similar in amino acid sequence to the flexible pilin of Aeromonas hydrophila. These results have important implications in the construction of stable, live attenuated cholera vaccines.