Mucus plugging is a hallmark of cystic fibrosis, but the link between the defective gene product, the cystic fibrosis transmembrane conductance regulator, and the abnormal mucus phenotype is unclear. To demonstrate CFTR involvement in mucin glycoprotein secretion in epithelial cells, a retroviral vector was used to overexpress CFTR in gallbladder epithelial cells, and constitutive mucin labeling and secretion were monitored. Achievement of high-level vector expression was confirmed by transduction with marker genes. Cells transduced with vectors carrying CFTR cDNA showed 5-fold increased expression of CFTR by Western blotting. Mucin labeling and secretion were 4-fold elevated in transduced cells. These results suggest constitutive mucin synthesis and secretion in gallbladder epithelial cells are regulated by CFTR.