Cystic fibrosis transmembrane conductance regulator (CFTR) has been found to be colocalized with G-protein-coupled receptors (GPCRs) and the downstream signaling molecules; however, the mechanisms of the colocalization remain largely elusive. The present work has investigated the role of lipid rafts in the localized signaling from GPCRs to CFTR. Using commonly used sucrose gradient centrifugation, we found that CFTR along with G(alpha)S was associated with lipid rafts, and the association was disrupted by cholesterol depletion with methyl-beta-cyclodextrin (MCD) treatment in Calu-3 human airway epithelial cells. Using short-circuit current (I (sc)) as a readout of CFTR in Calu-3 cells or T84 human colonic epithelial cells, we showed that MCD, while increasing basal membrane permeability, had no effect on the I (sc) induced by several GPCR agonists. Similar results were also obtained with a cholesterol biosynthesis inhibitor lovastatin and a cholesterol-binding agent filipin in Calu-3 cells. Furthermore, cholesterol depletion did not impair cyclic AMP production elicited by the GPCR agonists in Calu-3 cells. Our data suggest that GPCR-mediated signaling maintain their integrity after lipid raft disruption in Calu-3 and T84 epithelial cells and cast doubts on the role of lipid rafts as signaling platforms in GPCR-mediated signaling.