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The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP binding cassette (ABC) transporter that functions as a phosphorylationand nucleotide-regulated chloride channel, is mutated in cystic fibrosis (CF) patients. Deletion of a phenylalanine at amino acid position 508 (⌬F508) in the first nucleotide binding domain (NBD1) is the most prevalent CF-causing mutation and results in defective protein processing and reduced CFTR function, leading to chloride impermeability in CF epithelia and heterologous systems. Using a STE6/CFTR⌬F508 chimera system in yeast, we isolated two novel ⌬F508 revertant mutations, I539T and G550E, proximal to and within the conserved ABC signature motif of NBD1, respectively. Western blot and functional analysis in mammalian cells indicate that mutations I539T and G550E each partially rescue the CFTR⌬F508 defect. Furthermore, a combination of both revertant mutations resulted in a 38-fold increase in CFTR⌬F508-mediated chloride current, representing 29% of wild type channel activity. The G550E mutation increased the sensitivity of CFTR⌬F508 and wild type CFTR to activation by cAMP agonists and blocked the enhancement of CFTR⌬F508 channel activity by 2 mM 3-isobutyl-1-methylxanthine. The data show that the ⌬F508 defect can be significantly rescued by second-site mutations in the nucleotide binding domain 1 region, that includes the LSGGQ consensus motif. Cystic fibrosis (CF)1 is the most frequent lethal genetic disease associated with a single gene in Caucasians (1). CF results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an ATP binding cassette (ABC) transporter that functions as a phosphorylation and nucleotide-regulated chloride channel located in the apical membrane of epithelial cells (2, 3). The ABC transporters constitute a large family of ubiquitously expressed proteins, mostly involved in ATP-driven translocation of diverse substrates across biological membranes (4, 5). It has been proposed that a functional ABC transporter has a minimal structural requirement of two membrane-spanning domains and two nucleotide binding domains (NBDs) (5). The NBDs, or ABC cassettes, share 30 -50% sequence identity (6) and are characterized by the presence of three conserved motifs; Walker A and Walker B motifs are present in several nucleotide binding and hydrolyzing proteins (7), and the ABC-signature motif, located just upstream of the Walker B, is diagnostic of ABC cassettes (5, 6).The deletion of the Phe-508 (⌬F508) in the first nucleotide binding domain (NBD1) of CFTR is the most frequent CFcausing mutation, present in 90% of CF chromosomes. ⌬F508 impairs normal protein maturation and trafficking to the plasma membrane (8, 9), presumably through a localized effect on the folding of the NBD1 domain (10 -12). This misfolding results in retention of CFTR⌬F508 by the endoplasmic reticulum-associated quality control and in subsequent degradation with the participation of the cytoplasmic proteasome (13). The CFTR...