The most common cystic fibrosis mutation deletes phenylalanine 508 in CFTR (CFTR-F508). This mutation causes the loss of CFTR Cl-channel activity by disrupting biosynthetic processing so that mutant protein does not reach the plasma membrane. It also decreases the rate at which mutant channels open. To identify second-site mutations that could reverse the effects of delta F508, we used a chimeric yeast STE6/CFTR system bearing the delta F508 mutation. This chimera manifests defective mating. After mutagenesis of the first nucleotide-binding domain, we found that mutation R555K partially restored mating. However, it also increased mating in the chimera lacking the delta F508 mutation. When we introduced the R555K mutation into human CFTR-delta F508, we found that the loss of apical Cl-current caused by delta F508 was partially restored, predominantly due to a partial correction of the delta F508 processing defect. Analysis of single CFTR Cl-channels showed that the R555K mutation did not correct the prolonged closed time associated with delta F508, rather it increased activity of both wild-type and delta F508 channels by prolonging the duration of bursts of activity. These data suggest that the region around residue R555 in the first nucleotide-binding domain is important both in determining the ability of the delta F508 protein to be properly processed and in determining channel function.