Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel with complex regulation. To explore CFTR structure and function, we exploited functional differences between human and murine CFTR. When compared with human CFTR, murine CFTR has a reduced single-channel conductance (human, 8.29 ± 0.18 pS; murine, 5.76 ± 0.16 pS; [1]) and a distinct pattern of channel gating, characterised by prolonged residence in a sub-conductance state [2]; transitions to sub-conductance states are rare for human CFTR. In previous work, we demonstrated that transfer of both NBDs of murine CFTR endowed human CFTR with dramatically prolonged channel openings similar to those of the sub-conductance state of murine CFTR [3]. However, transfer of both NBDs failed to endow human CFTR with a sub-conductance state like that of murine CFTR [3]. To investigate whether the membrane-spanning domains (MSDs) define the sub-conductance state of murine CFTR, we constructed the human-murine CFTR (hmCFTR) chimeras hmTM1-6, hmTM7-12 and hmTM1-6:TM7-12 containing MSD1, MSD2 and MSD1+2 of murine CFTR and studied their single-channel behaviour in excised inside-out membrane patches. All hmCFTR chimeras exhibited a hybrid gating pattern intermediate between that of human and murine CFTR. Reminiscent of murine CFTR, channel openings were very flickery with open probability decreasing in the rank order: human CFTR > hmTM7-12 > hmTM1-6 > hmTM1-6:TM7-12. The single-channel current amplitude of the full open state of hmCFTR chimeras was smaller than that of human CFTR, decreasing in the rank order: human CFTR > hmTM7-12 > hmTM1-6 ≥ hmTM1-6:TM7-12. Like human CFTR, hmTM7-12 opened predominantly to the full open state; transitions to sub-conductance states were unusual. However, hmTM1-6 resided in multiple sub-conductance states, which were visualised in heavily filtered single-channel recordings and current amplitude histograms. Of note, like murine CFTR, hmTM1-6:TM7-12 resided in a tiny sub-conductance state and only sojourned infrequently to the full open-state. Consistent with hmTM1-6:TM7-12 possessing human NBDs, this construct resided in the sub-conductance state for much shorter periods than murine CFTR. Taken together, our data suggest that amino acid sequences from both MSDs specify the sub-conductance state of murine CFTR and determine intraburst gating.