Proline residues located in membrane spanning domains of transport proteins are thought to play an important structural role, In the cystic fibrosis transmembrane conductance regulator (CFTR), the predicted transmembrane segments contain four prolines: Pro(99), Pro(205), Pro(324), and Pro(1021). These residues are conserved across species, and mutations of two (P99L and P205S) are associated with cystic fibrosis, To evaluate the contribution of these prolines to CFTR Cl- channel function, we mutated each residue individually to either alanine or glycine or mutated all four simultaneously to alanine (P-Quad-A), We also constructed the two cystic fibrosis-associated mutations, cAMP agonists stimulated whole cell Cl- currents in HeLa cells expressing the individual constructs that resembled those produced by wild-type CFTR, However, the amount of current was decreased in the rank order: wild-type CFTR = Pro(324) > Pro(1021) > Pro(99) greater than or equal to Pro(205) mutants, The anion selectivity sequence of the mutants (Br- greater than or equal to Cl- > I-) resembled wild-type except for P99L (Br- greater than or equal to Cl- = I-). Although the Pro(99), Pro(324) , and Pro(1021) mutants produced mature protein, the amount of mature protein was much reduced with the pro205 mutants, and the P-Quad-A made none. Because the Pro(99) constructs produced mature protein but had altered whole cell currents, we investigated their single-channel properties, Mutant channels were regulated like wild-type CFTR; however, single-channel conductance was decreased in the rank order: wild-type CFTR greater than or equal to P99G > P99L greater than or equal to P99A. These results suggest that proline residues in the transmembrane segments are important for CFTR function, Pro(205) is critical for correct protein processing, and Pro(99) may contribute either directly or indirectly to the Cl- channel pore.