Acute adenosine 3',5'-cyclic monophosphate (cAMP) stimulation of intestinal epithelium induces net transepithelial Cl- secretion and inhibits neutral coupled NaCl absorption. To investigate the role that the cystic fibrosis transmembrane conductance regulator (CFTR) plays in these events, we measured bioelectric changes and radioisotopic NaCl flux across jejunal tissues from gene-targeted cftr "knockout" mice [cftr(-/-) homozygotes] and their normal littermates [cftr(+/+) homozygotes and cftr(+/-) heterozygotes]. Before stimulation, the short-circuit current (Isc, an index of Cl- secretion) of the cftr(-/-) jejunum was essentially zero and significantly less than in the cftr(+/+) or cftr(+/-) intestine. Acute cAMP stimulation had little effect on the bioelectric parameters of the cftr(-/-) intestine but induced a marked increase of Isc and decrease of total tissue conductance in both the cftr(+/+) and cftr(+/-) intestine. Differences in the magnitude of the cAMP-induced Isc between the cftr(+/+) and cftr(+/-) intestine were only observed when the cell-to-lumen anion concentration gradient was maximized by removal of permeant anions from the luminal bath. Radioisotope flux measurements revealed that Na+ and Cl- were absorbed equally across the cftr(-/-) jejunum under basal conditions. In cftr(+/+) and cftr(+/-) intestine, Na+ was absorbed at a similar rate, but net Cl- absorption was reduced from that in cftr(-/-) intestine by an amount approximating the Isc. Acute cAMP stimulation of the cftr(+/+) and cftr(+/-) intestine abolished net NaCl absorption and induced electrogenic Cl- secretion. In contrast, net NaCl absorption was unchanged from the preceding flux period in the cftr(-/-) jejunum. The data suggest that CFTR not only mediates cAMP-induced transepithelial Cl- secretion but is also required for cAMP inhibition of neutral NaCl absorption in the intestine.