Vibrio cholerae causes the potentially lethal disease cholera through the elaboration of the intestinal secretogen cholera toxin. A second toxin of V. cholerae, Zot, decreases intestinal tissue resistance by modifying intercellular tight junctions. In this report, a third toxin of V. cholerae, Ace (accessory cholera enterotoxin), is described. Ace increases short-circuit current in Ussing chambers and causes fluid secretion in ligated rabbit ileal loops. The predicted protein sequence of Ace shows striking similarity to eukaryotic ion-transporting ATPases, including the product of the cystic fibrosis gene. The gene encoding Ace is located immediately upstream of the genes encoding Zot and cholera toxin. The ctx, zot, and ace genes, which are located on a dynamic sector of the chromosome, comprise a V. cholerae "virulence cassette."