Mutations in the membrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) are the cause of CF disease. To determine how these various mutations alter CFTR structure and function, we have performed SDS-PAGE shift analysis, circular dichroism, and tyrosine fluorescence spectroscopy on wild type and CF-phenotypic mutants of the transmembrane (TM) hairpin of CFTR TM9-10. Various interhelical interactions were detected: mutants A 1006E and V 1008D's non-native hydrogen bond potential partner were confirmed respectively; and a salt bridge and hydrogen bond network was elucidated among R 1030, D993 and Y 1032. Studies of more than 20 mutant CFTR TM9-10 hairpin constructs revealed that the method of SDS-PAGE gel shift analysis is more sensitive to the removal of interhelical interactions near the loop region of TM hairpins than to other TM positions. The results show that various CF-phenotypic mutants can significantly alter the wt hairpin structure, and thereby produce aberrant CFTR function.