Cystic fibrosis (CF) is a frequent autosomal recessive disorder caused by mutation of a gene encoding a multifunctionnal transmembrane protein located in the apical membrane of epithelial cells lining exocrine glands, the Cystic Fibrosis Transmembrane conductance Regulator (CFTR). In an attempt to get a more complete picture of the pleiotropic effects of the CFTR defect on epitelial cells and particularly on the membrane compartment, a bi-dimensional BN/SDS PAGE-based proteomic approach was used on colonic crypts samples from control and CFTR-knockout mice (cftr^-/-). This approach overcomes the difficulties of membrane protein analysis by conventional 2-D PAGE and is able to resolve multiprotein complexes. Used here for the first time on crude membrane proteins that were extracted from murine colonic crypts, BN/SDS PAGE allows effective separation of protein species and complexes of various origins, including mitochondria, plasma membrane, and intracellular compartments. The major statistically significant difference in protein maps obtained with samples from control and cftr^-/- mice was unambiguously identified as mClCA3, a member of a family of calcium-activated chloride channels (CaCCs), considered as key molecules in mucus secretion by goblet cells. On the basis of this finding, we evaluated the overall expression and localization of mClCA3 in the colonic epithelium and in the lung of mice by immunoblot analysis and immunohistochemistry. We found that mClCA3 expression was significantly decreased in the colon and lung of the cftr^-/- mice. In an ex-vivo assay, we show that the Ca²⁺-dependent (carbachol-stimulated) glycoprotein secretion strongly inhibited by the CaCCs blocker, niflumic acid (100µM), is impaired in the distal colon of cftr^-/- mice. These results support the conclusion that a ClCA-related function in the CF colon depends on CFTR expression and may be correlated with the impaired expression of mClCA3.