Two phenotypic missense mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel pore [L346P and R347P in transmembrane (TM) segment 6] involve gain of a proline residue, but only L346P represents a significant loss of segment hydropathy. We show here that for synthetic peptides corresponding to sequences of CFTR TM6 segments, circular dichroism spectra of wild type and R347P TM6 in membrane mimetic environments are virtually identical, but L346P loses ca. 50% helicity – implying a membrane insertion defect in the latter mutant. A similar defect was observed in the corresponding double-spanning (‘hairpin’) TM5/6-L346P synthetic peptide. Examination of the biogenesis of CFTR revealed that the full-length protein harboring the L346P mutation is rapidly degraded at the endoplasmic reticulum (ER), while the wild type and the R347P protein process normally. Furthermore, a second site mutation (R347I) that restores in vitro membrane insertion and folding of the TM5/6-L346P peptide also rescues the folding and cell surface chloride channel function of full-length L346P CFTR. The correlated in vitro/in vivo results demonstrate that destabilizing local hydrophobic character represents a sufficient signal for marking CFTR as a non-native protein by the ER quality control, with accompanying deleterious consequences to global protein folding events.