A powerful approach to investigate ion channel structure-function relationships is to exploit functional differences between orthologues from divergent species.  Here, this approach is used to study the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR).  Mouse CFTR has 79% shared amino acid identity with human CFTR.  Its function differs from human CFTR in several important respects: altered gating pattern distinguished by prolonged residence in a sub-conductance state, reduced single-channel conductance and strong inward rectification (1,2).  Transfer of the regulatory (R) domain of mouse CFTR to human CFTR was without effect, whereas transfer of both nucleotide-binding domains (NBDs) endowed human CFTR with greatly prolonged channel openings without conferring human CFTR with the sub-conductance state, single-channel conductance and inward rectification of mouse CFTR (3).  To test the idea that the membrane-spanning domains (MSDs) govern the conduction properties of human and murine CFTR, the human-murine CFTR (hmCFTR) chimeras hmTM1-6, hmTM7-12 and hmTM1-6:TM7-12 containing MSD1, MSD2 and MSD1+MSD2 of mouse CFTR on a human CFTR background were synthesized.  To study the single-channel behaviour of these hmCFTR chimeras, a large Cl^– concentration gradient ([Cl^–]_int, 147 mM; [Cl^–]_ext, 10 mM) was imposed across inside-out membrane patches excised from transiently transfected CHO cells and voltage clamped at -50 mV, whereas to analyse macroscopic currents, membrane patches were bathed in symmetrical 147 mM Cl^– solutions and voltage clamped at 0 mV.  All intracellular solutions contained protein kinase A (PKA; 75 nM) and ATP (1 mM) to activate and sustain CFTR channel activity; temperature was 37 °C.  The three hmCFTR chimeras possessed a gating pattern intermediate between those of human and mouse CFTR.  Like human CFTR, hmTM7-12 rarely transitioned to sub-conductance states, whereas hmTM1-6 sojourned to multiple sub-conductance states and hmTM1-6:TM7-12 resided in a tiny sub-conductance state resembling that of mouse CFTR.  Consistent with hmTM1-6:TM7-12 possessing human, not mouse, NBDs, this hmCFTR chimera resided in the sub-conductance state for noticeably shorter periods than mouse CFTR.  Upon quantification, both the single-channel conductance and open probability of the full open-state of hmCFTR chimeras decreased in the rank order: human CFTR > hmTM7-12 > hmTM1-6 ≥ hmTM1-6:TM7-12 (n = 5 – 10).  Using 2-s voltage ramps from -100 to +100 mV to acquire current-voltage relationships, human CFTR Cl^– currents weakly inwardly rectified, whereas those of mouse CFTR exhibited strong inward rectification.  Transfer of both MSDs of mouse CFTR to human CFTR in the chimera hmTM1-6:TM7-12 reproduced the strong inward rectification of mouse CFTR (n = 4).  Interestingly, the inward rectification of hmTM1-6 closely resembled that of mouse CFTR, whereas that of hmTM7-12 was intermediate between human and mouse CFTR (n = 6 – 9).  Taken together, these data demonstrate that sequences from both MSDs specify the gating, conduction and rectification properties of CFTR with sequences from MSD1 playing a dominant role in determining the different rectification behaviour of human and mouse CFTR.