The antisecretory effect of berberine sulfate (BS) was determined in the rabbit ileal mucosa in vitro mounted in Ussing chambers. When present in the luminal side, the drug had no effect on potential difference (PD) or on the short-circuit current (Isc) changes induced by theophylline or prostaglandin E2 (PGE2). When present in the serosal medium at a concentration of 2 mM, however, berberine reduced PD and Isc and abolished residual ion flux (bicarbonate secretion) but had no significant affect on basal net Na and Cl transepithelial transport. BS effect on basal PD was not affected by the absence of Ca2+ in the medium; neither was it inhibited by the alpha-adrenergic blocker yohimbine. Serosally added BS effectively antagonized the secretory effect on Isc of agents that increase each of the known intracellular mediators of ion secretion: cyclic 3',5'-adenosine monophosphate (cAMP), cyclic 3',5'-guanosine monophosphate (cGMP), and Ca2+; it also inhibited the ion transport modifications evoked by PGE2. The drug had no effect on the concentration of cyclic nucleotides either under basal condition or in the presence of some secretagogues. Finally, BS did not impair the PD rise due to luminal addition of glucose.