VX-770 (Ivacaftor) has been approved for clinical usage in CF patients with several CFTR mutations. Yet the binding site(s) on CFTR for this compound and other small molecule potentiators are unknown. We hypothesize that insight into this question could be gained by comparing the effect of potentiators on CFTR channels from different origins, e.g., human, mouse and Xenopus (frog). In the present study, we combined this comparative molecular pharmacology approach with that of computer-aided drug discovery to identify and characterize new potentiators of CFTR, and to explore possible mechanism of action. Our results demonstrate that: (1) VX-770, NPPB, GlyH-101, P1, P2, and P3 all exhibited ortholog-specific behavior in that they potentiated hCFTR, mCFTR, and xCFTR with different efficacies; (2) P1, P2, and P3 potentiated hCFTR in excised macropatches in a manner dependent upon the degree of PKA-mediated stimulation; (3) P1 and P2 did not have additive effects, suggesting that these compounds might share binding sites; and (4) Using a pharmacophore modeling approach, we identified three new potentiators (IOWH-032, OSSK-2, and OSSK-3) that have structures similar to GlyH-101 and that also exhibit ortholog-specific potentation of CFTR. These could potentially serve as lead compounds for development of new drugs for the treatment of cystic fibrosis. The ortholog-specific behavior of these compounds suggest that a comparative pharmacology approach, using cross-ortholog chimeras, may be useful for identification of binding sites on human CFTR.