Misfolding accounts for the endoplasmic reticulum–associated degradation of mutant cystic fibrosis transmembrane conductance regulators (CFTRs), including deletion of Phe508 (ΔF508) in the nucleotide-binding domain 1 (NBD1). To study the role of Phe508, the de novo folding and stability of NBD1, NBD2 and CFTR were compared in conjunction with mutagenesis of Phe508. ΔF508 and amino acid replacements that prevented CFTR folding disrupted the NBD2 fold and its native interaction with NBD1. ΔF508 caused limited alteration in NBD1 conformation. Whereas nonpolar and some aliphatic residues were permissive, charged residues and glycine compromised the post-translational folding and stability of NBD2 and CFTR. The results suggest that hydrophobic side chain interactions of Phe508 are required for vectorial folding of NBD2 and the domain-domain assembly of CFTR, representing a combined co- and post-translational folding mechanism that may be used by other multidomain membrane proteins.