ACE, accessory cholera enterotoxin, the third enterotoxin in Vibrio cholerae, has been reported to increase short-circuit current (I_sc) in rabbit ileum and to cause fluid secretion in ligated rabbit ileal loops. We studied the ACE-induced change in I_sc and potential difference (PD) in T84 monolayers mounted in modified Ussing chambers, an in vitro model of a Cl secretory cell. ACE added to the apical surface alone stimulated a rapid increase in I_sc and PD that was concentration dependent and immediately reversed when the toxin was removed. Ion replacement studies established that the current was dependent on Cl and HCO₃. ACE acted synergistically with the Ca²⁺-dependent acetylcholine analog, carbachol, to stimulate secretion in T84 monolayers. In contrast, the secretory response to cAMP or cGMP agonists was not enhanced by ACE. The ACE-stimulated secretion was dependent on extracellular and intracellular Ca²⁺ but was not associated with an increase in intracellular cyclic nucleotides. We conclude that the mechanism of secretion by ACE involves Ca²⁺ as a second messenger and that this toxin stimulates a novel Ca²⁺-dependent synergy.