Peptide toxins have been valuable probes in efforts to identify amino acid residues that line the permeation pathway of cation-selective channels. However, no peptide toxins have been identified which interact with known anion-selective channels such as CFTR. CFTR channels are expressed in epithelial cells and are associated with a number of genetic disorders, including cystic fibrosis and polycystic kidney disease. Several organic inhibitors have been used to investigate the structure of the chloride permeation pathway in CFTR. However, investigations of the wider cytoplasmic vestibule have been hindered due to the absence of a high affinity blocker that interacts with residues in this area. In this study we show that venom of the scorpion L. quinquestriatus hebraeus reversibly inhibits CFTR, in a voltage-independent manner, by decreasing the single channel mean burst duration and open probability only when applied to the cytoplasmic surface of phosphorylated channels. Venom was able to decrease burst duration and open probability even when CFTR channels were locked open by treatment with either vanadate or 5'-adenosine (, -imino) triphosphate, and block was strengthened upon reduction of [Cl^-]_o, suggesting inhibition by a pore-block mechanism. Venom had no effect on ATP-dependent macroscopic opening rate in channels studied by inside-out macropatches. Interestingly, the inhibitory activity was abolished by proteinase treatment. We conclude that a peptide toxin contained in the scorpion venom inhibits CFTR channels by a pore block mechanism; these experiments provide the first step toward isolation of the active component, which would be highly valuable as a probe for CFTR structure and function.