Chemical modification can inhibit ion channels either by reacting with pore-lining residues and directly occluding the channel or by closing the channel allosterically. A general method to distinguish between these two mechanisms does not exist. Previously, sulfhydryl (SH) modification has been shown to inhibit ATP-sensitive K(+) (K(ATP)) channels. The crucial modification has been localized to C42 near the N-terminus of Kir6.2, a pore-forming subunit of K(ATP) channels, but little is known about how SH modification of C42 causes channel inhibition. To investigate this mechanism, we used the membrane-impermeable methanethiosulfonates, MTSET and MTS-TEAH, to modify Kir6.2 channels. While intracellular application of MTSET irreversibly inhibited channels, MTS-TEAH failed to do so. Instead, MTS-TEAH treatment prolonged channel openings and prevented the effect of subsequent MTSET treatment. Similar observations were made in mutants in which cysteines other than C42 had been mutated. Neither MTSET nor MTS-TEAH, however, affected mutant channels in which valines were substituted for C42 residues in all subunits. The reagents were effective when two of four C42 residues in the tetramer were replaced by valines. These results can be interpreted as indicating that both reagents modify C42. We then employed spermine, a known inner pore blocker, as a probe to examine whether MTS-TEAH modification alters pore accessibility. We found that spermine block was not changed by MTS-TEAH modification. Based on these data, we postulate that C42 faces either the cytoplasm or a vestibule section wide enough to allow spermine to pass freely after modification by MTS-TEAH. Our study suggests that channel inhibition caused by SH modification of Kir6.2 is an allosteric effect, and is not caused by direct pore blockage.