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To explore the relationship between structure and function in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel, we studied XenopusCFTR. We found that the anion permeability sequence of cAMP-activated Cl-currents in the apical membrane of XenopusA6 epithelia differed from that of cAMP-activated Cl-currents in human epithelia expressing CFTR. To understand the molecular basis for this difference and to learn whether CFTR from another species would have properties similar to human CFTR, we assembled a full-length XenopusCFTR cDNA from A6 cells. Expression of XenopusCFTR in HeLa cells generated cAMP-activated whole-cell currents and cAMP-dependent protein kinase-activated single channels that resembled those of human CFTR with the exception that the anion permeability sequence was different (Br-= I-> Cl-in XenopusCFTR and Br-= Cl-> I-in human). In addition, the single-channel conductance of XenopusCFTR was increased. To investigate protein regions that account for these differences, we constructed chimeric proteins by replacing either the first or second membrane-spanning domain of human CFTR with the equivalent region of XenopusCFTR (hX1-6 and hX7-12, respectively) and examined their function in HeLa cells. We found that the anion permeability sequence (Br-= I-> Cl-) and single-channel conductance of hX1-6 resembled that of XenopusCFTR expressed in HeLa cells, whereas hX7-12 had properties like those of human CFTR. However, the gating of hX1-6 showed a flickery behavior. The altered gating of hX1-6 was attributed to residues in the first extracellular loop of XenopusCFTR because mutation of residues in that region to the corresponding residues of human CFTR produced gating behavior similar to that of human CFTR. These data suggest that sequence differences in the first membrane-spanning domains are responsible for the differences in the permeation properties of human and XenopusCFTR and that the first extracellular loop influences channel gating.