The major disease-causing mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine 508 (F508), which adversely affects processing and plasma membrane targeting of CFTR. Under conditions predicted to stabilize protein folding, F508 CFTR is capable of trafficking to the plasma membrane and retains cAMP-regulated anion channel activity. Overexpression is one factor that increases CFTR trafficking; therefore, we hypothesized that expression of a domain mimic of the first nucleotide-binding fold of CFTR, i.e., the site of F508, may be sufficient to overwhelm the quality control process or otherwise stabilize F508 CFTR and, thereby, restore cAMP-stimulated anion secretion. In epithelial cells expressing recombinant F508 hCFTR, expression of wild-type NBF1 (NBF1) increased the amount of both core-glycosylated and mature protein to a greater extent than did expression of F508 NBF1. Expression of NBF1 in the F508 hCFTR cells increased whole cell chloride current density to ~50% of that in cells expressing wild-type hCFTR. Expression of NBF1 in polarized epithelial monolayers from a F508/F508 cystic fibrosis mouse (MGEF) restored cAMP-stimulated transepithelial anion secretion, but not in monolayers from a CFTR-null mouse (MGEN). Restoration of anion secretion was sustained in NBF1-expressing MGEF for >30 passages whereas MGEN corrected with hCFTR progressively lost anion secretion capability. We conclude that expression of a NBF1 domain mimic may be useful for correction of the F508 CFTR protein trafficking defect in cystic fibrosis epithelia.