Vacuolar ATPases (V-ATPases) are highly conserved proton pumps that regulate organelle pH. Epithelial luminal pH is also regulated by cAMP-dependent traffic of specific subunits of the V-ATPase complex from endosomes into the apical membrane. In the intestine, cAMP-dependent traffic of cystic fibrosis transmembrane conductance regulator (CFTR) channels and the sodium hydrogen exchanger (NHE3) in the brush border regulate luminal pH. V-ATPase was found to co-localize with CFTR in intestinal CFTR High Expresser (CHE) cells recently. Moreover, apical traffic of V-ATPase and CFTR in rat Brunner's glands was shown to be dependent on cAMP/PKA. These observations support a functional relationship between V-ATPase and CFTR in the intestine. The current study examined V-ATPase and CFTR distribution in intestines from wild-type, CFTR^-/- mice and polarized intestinal CaCo-2 _BBe cells following cAMP stimulation and inhibition of CFTR/V-ATPase function. Co- immunoprecipitation studies examined V-ATPase interaction with CFTR. The pH sensitive dye BCECF determined proton efflux and its dependence on V-ATPase/CFTR in intestinal cells. cAMP increased V-ATPase/CFTR co-localization in the apical domain of intestinal cells, and redistributed the V-ATPase V_oa1 and V_oa2 trafficking subunits from the basolateral membrane to the BBM. V_oa1 and V_oa2 subunits were localized to endosomes beneath the terminal web in untreated CFTR ^-/- intestine, but redistributed to the subapical cytoplasm following cAMP treatment. Inhibition of CFTR or V-ATPase significantly decreased pH_i in cells, confirming their functional interdependence. These data establish that V-ATPase traffics into the brush border membrane to regulate proton efflux and this activity is dependent on CFTR in the intestine.