Studies of widely diverse species of a protein are a powerful tool to gain information on the structure and function of the protein. We investigated the response of human, pig, shark and killifish cystic fibrosis trans-membrane conductance regulator (CFTR) to specific inhibitors of the channel: CFTRinh-172, GlyH-101, and glibenclamide. In several expression systems, including isolated perfusions of the rectal gland, primary cell cultures of rectal gland tubules and oocyte expression, we observed fundamental differences in the sensitivity to inhibition by these CFTR blockers. We used primarily two-electrode voltage clamping of cRNA microinjected Xenopus laevis oocytes. In oocyte studies, shark CFTR was insensitive to CFTRinh-172 (maximum inhibition 8 ± 1.4% at 20µM), pCFTR was insensitive to Glibenclamide (maximum inhibition 12.8 ± 4.2% at 200µM), and all species were sensitive to GlyH-101 (maximum inhibition with pCFTR of 80.2 ± 3.6% at 20µM). Shark CFTR was completely insensitive to inhibition by CFTRinh-172 in short circuit current experiments (2.5 ± 0.15 % inhibition of chloride secretion) compared to inhibition with GlyH-101 (56.5 ± 6.56 % inhibition of chloride secretion). Perfusion studies confirmed these results. These experiments demonstrate a profound difference in the sensitivity of different CFTR species to inhibition by CFTR blockers. However, the amino acid residues that have been proposed by site directed mutagenesis studies to be responsible for inhibitor binding are uniformly conserved in all four isoforms studied. Therefore, the differences cannot be explained by simply targeting one amino acid for site-directed mutagenesis. Rather, the potency of the inhibitory actions of CFTRinh-172, Gly-H101 and glibenclamide on the CFTR molecule is dictated by the local environment and the three dimensional structure of residues that form the vestibule and the chloride pore.