Defective trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common cause of cystic fibrosis. In chloride- secreting epithelia, it is well established that CFTR localizes to intracellular organelles and to apical membranes. However, it is controversial whether secretagogues regulate the trafficking of CFTR. To investigate whether acute hormonal stimulation of chloride secretion is coupled to the trafficking of CFTR, we used the intact shark rectal gland, a model tissue in which salt secretion is dynamically regulated and both chloride secretion and cellular CFTR immunofluorescence can be quantified in parallel. In rectal glands perfused under basal conditions without secretagogues, C1- secretion was 151±65 μeq/h/g. Vasoactive intestinal peptide (VIP), forskolin, and genistein led to 10-, 6-, and 4-fold increases in C1- secretion. In basal glands, quantitative confocal microscopy revealed CFTR immunofluorescence extending from the apical membrane deeply into the cell (7.28±0.35 μm). During stimulation with secretagogues, apical extension of CFTR immunofluorescence into the cell was reduced significantly to 3.24±0.08 μm by VIP, 4.08±0.13 by forskolin, and 3.19±0.1 by genistein (P <0.001). Moreover, the peak intensity of CFTR fluorescence shifted towards the apical membrane (peak fluorescence 2.5±0.13 μm basal vs. 1.51±0.06, 1.77±0.1, and 1.38±0.05 for VIP, forskolin, and genistein; all P < 0.001). The increase in both C1- secretion and apical CFTR trafficking reversed to basal values after removal of VIP. These data provide the first quantitative morphological evidence for acute hormonal regulation of CFTR trafficking in an intact epithelial tissue.