E1/E3-deleted adenoviral vectors expressing an N-terminal green fluorescent protein (GFP) reporter gene fused to either wtCFTR (H5.040CMVEGFP-wtCFTR) or ΔF508-CFTR (H5.040CMVEGFP-ΔF508CFTR) were generated. To characterize the expression and activity, A549 cells were infected with vectors expressing GFP-tagged and non-tagged forms of CFTR and ΔF508CFTR. CFTR activity was assayed in cell-attached and excised patches. For H5.040CMVEGFP-wtCFTR, forskolin-dependent outward current was observed in cell-attached patches from 56 of 67 GFP-positive cells. Single-channel conductances, open probability, mean open and mean closed time values for GFP-CFTR and CFTR were not significantly different. After excision, GFP-CFTR activity required ATP and exhibited a linear I-V relationship. For H5.040CMVEGFP-ΔF508CFTR, media were supplemented with 5 mM butyrate 16 h after infection. Forskolin-dependent outward current was observed in cell-attached patches from 21 of 30 butyrate-treated GFP-positive cells and 0 of 8 GFP-positive cells without butyrate. Single-channel conductances, open probability, mean open and mean closed time values for GFP-ΔF508CFTR and ΔF508CFTR were not significantly different. However, the increase in open probability with genistein was significantly smaller for GFP-ΔF508CFTR than for ΔF508CFTR. In excised patches, GFP-ΔF508CFTR activity required ATP and exhibited a linear I-V relationship. Despite the consistent detection of GFP-CFTR and GFP-ΔF508CFTR channels in the plasma membrane by patch clamping, GFP fluorescence was observed only in intracellular regions and was not altered by butyrate. The data show that high levels of functional GFP-tagged CFTR channels can be expressed with these adenoviral vector constructs.