The structural basis of general anesthesia has remained hypothetical. Ion channels are plausible targets, as general anesthetics interact directly with neuronal ion channels and modulate their function. Investigating these interactions at the molecular level will further the understanding of general anesthesia and aid in the design of safer and more effective general anesthetics. The voltage-gated K+ channel Shaw2 is inhibited by relevant doses of n-alcohols and inhaled anesthetics at a discrete site. To map the residues that constitute this site in the activation gate of the channel, we conducted alanine-scanning mutagenesis in the S4-S5 linker and the S6 segment (post PVPV) of the Shaw2 protein and expressed the resulting mutants in Xenopus oocytes. Characterization of the 1-butanol and halothane dose-inhibition relations under voltage-clamp conditions and double mutant cycle analysis revealed significant energetic effects of single and double mutations on the inferred drug binding. Double mutants, such as Q320A/Y420A and A326V/Y419A (between the S4-S5 and S6), and Q320A/A326V (within the S4-S5 linker), exhibited the following coupling coefficients: 1.6, 4.3 and 2.8, respectively. Additional mutations suggested that these effects are not the result of altered gating. Exploiting these results, molecular dynamics simulations and docking calculations demonstrate the structural contributions of the S4-S5 linker and the distal S6 segment to a putative amphiphilic cavity that binds general anesthetics in the Shaw2 channel. Photolabeling of the Shaw2 protein is currently being explored as a method to test the hypothesis that this cavity is an anesthetic binding site. In conclusion, this work provides compelling evidence for a molecular mechanism of general anesthesia in which inhaled anesthetics modulate function through direct binding to a discrete cavity in the ion channel protein. Supported by NIH T04324 (AF), CNPg 141009/2009-8 (CA), NIH GM55876 (RGE), NIH AA010615 (MC).