Cholera toxin secretion is dependent upon the extracellular protein secretion apparatus encoded by the eps gene locus of Vibrio cholerae. Although the eps gene locus encodes several type four prepilin-like proteins, the peptidase responsible for processing these proteins has not been identified. This report describes the identification of a prepilin peptidase from the V. cholerae genomic database by virtue of its homology with the PilD prepilin peptidase of Pseudomonas aeruginosa. Plasmid disruption or deletion of this peptidase gene in either EI Tor or classical V. cholerae O1 biotype strains results in a dramatic decrease in cholera toxin secretion. In the case of the EI Tor biotype mutants, surface expression of the type 4 pilus responsible for mannose-sensitive haemagglutination is abolished. The cloned V. cholerae peptidase processes either EpsI or MshA preproteins when co-expressed in E. coli. Mutation of the V. cholerae peptidase gene also results in a defect in virulence and decreased levels of OmpU. The V. cholerae peptidase gene sequence shows 80% homology with the Vibrio vulnificus VvpD type 4 prepilin peptidase required for pilus assembly and cytolysin secretion in V. vulnificus. Accordingly, the V. cholerae type 4 prepilin peptidase required for pilus assembly and cholera toxin secretion has been designated VcpD.