Peptide toxins are invaluable tools for studying the structure and physiology of ion channels. Pseudechetoxin (PsTx) is the first known peptide toxin that targets cyclic nucleotide-gated (CNG) ion channels, which play a critical role in sensory transduction in the visual and olfactory systems. PsTx inhibited channel currents at low nM concentrations when applied to the extracellular face of membrane patches expressing olfactory CNGA2 subunits. Surprisingly, 500 nM PsTx did not inhibit currents through channels formed by the CNGA3 subunit from cone photoreceptors. We have exploited this difference to identify the PsTx-binding site on the extracellular face of CNG channels. Studies using chimeric channels revealed that transplantation of the pore domain from CNGA2 was sufficient to confer high affinity PsTx binding upon a CNGA3 background. To further define the binding site, reciprocal mutations were made at 10 nonidentical amino acid residues in this region. We found that two residues in CNGA2, D316 and Y321, were essential for high-affinity inhibition by PsTx. Furthermore, replacement of both residues was required to confer high-affinity PsTx inhibition upon CNGA3. Several other residues, including E325, also form favorable interactions with PsTx. In the CNGA2-E325K mutant, PsTx affinity was reduced by approximately 5-fold to 120 nM. An electrostatic interaction with D316 does not appear to be the primary determinant of PsTx affinity, as modification of the D316C mutant with a negatively charged methanethiosulfonate reagent did not restore high affinity inhibition. The residues involved in PsTx binding are found within the pore turret and helix, in similar positions to residues that form the receptor for pore-blocking toxins in voltage-gated potassium channels. Furthermore, biophysical properties of PsTx block, including an unfavorable interaction with permeant ions, also suggest that it acts as a pore blocker. In summary, PsTx seems to occlude the entrance to the pore by forming high-affinity contacts with the pore turret, which may be larger than that found in the KcsA structure.