The cellular phenotype of the most common cystic fibrosis-causing mutation, deletion of phenylalanine 508 (deltaF508) in the amino-terminal nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR), is the inability of the mutant protein to fold and transit to the apical membrane of affected epithelial cells. Expressed NBD1s were purified and folded in vitro into soluble monomers capable of binding nucleotide. Here we report that the deltaF508 mutation has little effect on the thermodynamic stability of the folded NBD1. The deltaG(0)(D,GdnHCl) is 15.5 kJ/mol for the wild type NBD1 and 14.4 kJ/mol for NBD1deltaF. In contrast, the mutation significantly reduces the folding yield at a variety of temperatures, indicating that Phe-508 makes crucial contacts during the folding process, but plays little role in stabilization of the native state. Under conditions that approximate the efficiency of maturation in vivo, the rate off-pathway is significantly increased by the disease causing mutation. These results establish a molecular mechanism for most cases of cystic fibrosis and provide insight into the complex processes by which primary sequence encodes the three-dimensional structure.