Cystic fibrosis (CF) is caused by mutations in the gene encoding a chloride channel called the CF transmembrane conductance regulator (CFTR). A single mutation in this gene, deletion of three nucleotides that leads to the absence of phenylalanine 508 (i.e., ΔF508), is found on 70% of all CF chromosomes. To explore the molecular mechanism(s) responsible for defective chloride transport in patients with CF, we have studied the processing, localization, and function of wild type (W.T.), ΔF508 and G551D CFTR (a G→D missense mutation at position 551) in retrovirus transduced L cells. Cell transduced with W.T. CFTR expressed a 170 kd CFTR protein that was endoglycosidase H (Endo H) resistant, localized to the plasma membrane, and generated a cAMP-mediated anion conductance (GCl) when stimulated with standard concentrations of forskolin (5 μM), cpt cAMP (400 μM) and IBMX (100 μM). The G551D CFTR was indistinguishable from W.T. CFTR with respect to post-translational processing and localization, but it did not produce a cAMP-activated GCI in response to the standard stimulation cocktail. However, raising the IBMX concentration to 4 mM produced Gc, in G551D expressing cells. Cells transduced with ΔF508 CFTR expressed an Endo H sensitive CFTR protein (∼140 kd) that was found in a cytosolic, perinuclear location. These cells did not respond to the standard cocktail, but ∼20% of cells increased GCI when the cocktail contained 4 mM IBMX. Incubation of cells at 26°C for 48 hours prior to analysis elicited responses in ΔF508 expressing cells at low IBMX concentrations, but had no effect on the responses of cells expressing W.T. or G551D CFTR. The response of ΔF508 to 26°C was associated with plasma membrane localization of CFTR protein. These results suggest that there are two mechanisms whereby CFTR mutations lead to loss of cAMP-responsive GCI. First, shown by G551D CFTR, the protein can be processed and targeted to the plasma membrane correctly, but lack full responsiveness to stimulation by cAMP. Second, as examplified by ΔF508 CFTR, a partially functional protein which is not targeted to its correct cellular location can also lead to loss of the cAMP, responsive GCI.