The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel critical to intestinal anion secretion. In addition to phosphorylation, vesicle traffic regulates CFTR in some epithelial cells. Studies of cultured intestinal cells are conflicting regarding the role of cAMP-dependent vesicle traffic in regulating chloride transport. Whether CFTR is present in vesicular compartments within chloride secretory cells in the intestine is unknown and the role of cAMP-dependent vesicle insertion in regulating CFTR and intestinal fluid secretion remains unclear. The purpose of this study was to: (1) examine and quantify the subcellular distribution for CFTR in rat intestine, (2) further define the ultrastructure of the previously identified CFTR High Expresser (CHE) cell, and (3) examine the cellular distribution of CFTR following cAMP stimulation in vivo. Using the sensitive techniques of cryoimmunogold electron microscopy we identified CFTR in subapical vesicles and on the apical plasma membrane in crypt, Brunner glands, and CHE cells. cAMP stimulation in rat proximal small intestine produced a fluid secretory response and was associated with an apical redistribution of CFTR, supporting a physiologic role for cAMP-dependent CFTR vesicle insertion in regulating CFTR in the intestine.