The most common disease-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is the out-of-frame deletion of 3 nucleotides (CTT). This mutation leads to the loss of phenylalanine-508 (ΔF508) and a silent codon change (SCC) for isoleucine-507 (I507-ATC→ATT). AF508 CFTR is misfolded and degraded by endoplasmic reticulum-associated degradation (ERAD). We have demonstrated that the I507-ATC→ATT SCC alters ΔF508 CFTR mRNA structure and translation dynamics. By comparing the biochemical and functional properties of the I507-ATT and I507-ATC ΔF508 CFTR, we establish that the I507-ATC→ATT SCC contributes to the cotranslational misfolding, ERAD, and to the functional defects associated with ΔF508 CFTR We demonstrate that the I507-ATC ΔF508 CFTR is less susceptible to the ER quality-control machinery during translation than the I507-ATT, although 27°C correction is necessary for sufficient cell-surface expression. Whole-cell patch-clamp recordings indicate sustained, thermally stable cAMP-activated Cl- transport through I507-ATC and unstable function of the I507-ATT ΔF508 CFTR Single-channel recordings reveal improved gating properties of the I507-ATC compared to I507-ATT AF508 CFTR (NPo=0.45±0.037 vs. NPo=0. 09±0.002; P<0.001). Our results signify the role of the I507-ATC→ATT SCC in the AF508 CFTR defects and support the importance of synonymous codon choices in determining the function of gene products. © FASEB.