BACKGROUND: Vibrio cholerae gains natural competence upon growth on chitin. This allows the organism to take up free DNA from the environment and to incorporate it into its genome by homologous recombination. RESULTS: Making use of this developmental program in order to use it as a tool to genetically manipulate V. cholerae and potentially also others Vibrio species was envisaged. Therefore, we re-investigated the experimental design for natural transformation of V. cholerae and tested different donor DNA fragments with respect to their source (genomic versus PCR-derived), quantity, and homologous flanking regions. Furthermore, we simplified the procedure in terms of the chitin source used as inducer of natural competence and the composition of the growth medium. CONCLUSIONS: The current study allows us to recommend a standard protocol to genetically manipulate V. cholerae using commercially available sources of chitin and minimal medium, respectively, as well as PCR-derived donor DNA as transforming material.