The cellular origin of the response to secretagogues in small bowel epithelium was investigated by recording the effect of 5-hydroxytryptamine (5-HT, 10(-4) mol/l), acetylcholine (10(-4) mol/l), and prostaglandin E2 (PGE2, 10(-5) or 10(-4) mol/l) on apical membrane potentials (Va) of crypt and villus cells of rat ileum and jejunum in vitro using intracellular microelectrodes. Experiments were performed under visual control; addition of secretagogues and other manipulations were carried out during single impalements. Under basal conditions, apical membrane potential differences were consistently higher in jejunum than ileum (-72 +/- 1 vs. -47 +/- 2 mV, respectively, for villus impalements; -61 +/- 2 mV vs. -57 +/- 1 mV, respectively, for crypt impalements), and in jejunum villous membrane potentials exceeded those in the crypt. In the ileum, this crypt-villus gradient was reversed. The three secretagogues increased transmural potential difference and transiently reduced Va in cells in both crypt and villus regions by 8 mV or more. Fractional apical membrane resistance (FR) declined in ileum by approximately 30% in response to 5-HT and PGE2, whereas little change in FR was observed in jejunal recordings. PGE2 was ineffective in crypt and villus when Cl- was replaced by gluconate in both the luminal and serosal perfusates but depolarized the apical membrane in both regions after serosal restoration of Cl- from -76 +/- 4 to -56 +/- 8 mV in villus and from -58 +/- 4 to -45 +/- 6 mV in crypt. Rapid luminal Cl- substitution depolarized Va on the villus from -77 +/- 2 to -74 +/- 3 mV, but this effect was enhanced in the presence of PGE2, reducing Va from -65 +/- 8 to -43 +/- 12 mV. Prior to PGE2 addition, Va was -81 +/- 4 mV for this group of experiments. FR rose in the nominal absence of luminal Cl- from 0.69 +/- 0.09 to 0.77 +/- 0.06. It is concluded that because a Cl(-)-dependent depolarization of apical membrane potentials occurs in villi and crypts, net secretion in the small bowel is probably not confined to the crypts and may also occur from villous epithelium.