HT-29/B6 is a differentiated human colon carcinoma cell-line, whose clone accumulates polarized mucus-forming cell monolayer. This cell line natively expresses the cystic fibrosis transmembrane conductance regulator (CFTR) that responds to cAMP stimulation and mediates Cl- secretion. CFTR is mutated in the most frequent human genetic disease cystic fibrosis (CF), which is characterized by a reduced chloride secretion caused by the defect CFTR and a drastically increased Na+ absorption via the amiloride-sensitive epithelial sodium channel (ENaC). HT-29/B6 cells were grown to confluence on permeable filters and mounted into a modified Ussing chamber. Application of the CFTR activating cocktail of cAMP (100 mM) and IBMX (1 mM) increased the short-circuit current (ISC ), conductance (Gt) and capacitance (Ct). ISC and Gt could be reduced significantly using the Cl- channel blocker NPPB. The increase in capacitance shows that a prominent part of this activation is due to exocytotic insertion of preformed CFTR molecules into the apical membrane. Interestingly, this exocytotic insertion could be abolished in measurements using the PKA inhibitor H8. In another set of experiments we found that HT-29/B6 show no or tiny response to the ENaC blocker amiloride, although these cells constitutively express the a-ENaC subunit. Contrary to this, b- and [gamma]-ENaC subunits could not be detected on the mRNA level.