This study addresses the question of whether cholera toxin (CT) increases gut permeability for molecules greater than 3000 Da and whether such an effect is associated with an adjuvant function by CT on the gut immune response. We found that CT after oral administration gives rise to strikingly increased gut permeability for Dextran (Mw 3000) concomitantly with a strong enhancing effect on the anti-keyhole limpet hemocyanin (KLH) specific immune response in the lamina propria after oral immunization with KLH plus Dextran and CT. In contrast, the B-subunit of the holotoxin, which lacks the adenylate cyclase/cAMP-activating property of CT, failed to increase gut permeability as well as local anti-KLH immune responses. These results might suggest a causal linkage between the ability of CT to increase gut permeability and its adjuvant property on gut mucosal immune responses. In addition this finding supports the notion that the adenylate cyclase/cAMP system plays a regulatory role in gut permeability and is important in enhancing mucosal immune responses. Based on previous studies and the present data we propose that the mechanism for CT's adjuvant function on mucosal immune responses is by affecting antigen-presenting cells, T and B cells in the gut to give a net enhancing effect on the stimulation of local immunity, and that the CT-induced increase in gut permeability might be part of the adjuvant mechanism by facilitating luminal antigens to access the gut mucosal immune system.