To gain insight into the secondary structure of the ion conduction pathway of a voltage-gated K+ channel, we used sulfhydryl-specific reagents of different diameters to probe amino acid side-chain accessibilities in the pore of the channel after cysteine-substitution mutagenesis. We identified five positions at which modified amino acid side chains are accessible from the aqueous lumen of the external channel vestibule. Covalent coupling of the 2-trimethylammonium-thioethyl group to cysteine thiols leads to position-dependent current reduction, suggesting a gradual narrowing of the pore. The fact that the modified side chains of two adjacent amino acids are reactive is not compatible with the ion conduction pathway forming a regular beta-pleated sheet at these positions. The smaller thiol reagent Cd2+ reacts with modified side chains that are also accessible to the larger (2-trimethylammoniumethyl)methanethiosulfate (MTSET) [corrected]. Our results imply that the outer vestibule of a potassium-selective ion channel narrows over a short distance of three amino acids near a position where a regular beta-structure is unlikely.