The GABA_A receptor is a target of many general anesthetics. The low affinity of general anesthetics has complicated the search for the location of anesthetic binding sites. Attention has focused on two pairs of residues near the extracellular ends of the M2 and M3 membrane-spanning segments, α₁Ser270/β₂Asn265 (15′M2) and α₁Ala291/β₂Met286 (M3). In the 4-Å resolution acetylcholine receptor structure, the aligned positions are separated by ∼10 Å. To determine whether these residues are part of a binding site for propofol, an intravenous anesthetic, we probed propofol's ability to protect cysteines substituted for these residues from modification by the sulfhydryl-specific reagentp-chloromercuribenzenesulfonate (pCMBS^-). pCMBS^- reacted with cysteines substituted at the four positions in the absence and presence of GABA. Because propofol binding induces conformational change in the GABA_Areceptor, we needed to establish a reference state of the receptor to compare reaction rates in the absence and presence of propofol. We compared reaction rates in the presence of GABA with those in the presence of propofol +GABA. The GABA concentration was reduced to give a similar fraction of the maximal GABA current in both conditions. Propofol protected, in a concentration-dependent manner, the cysteine substituted for β₂Met286 from reaction with pCMBS^-. Propofol did not protect the cysteine substituted for the aligned α₁ subunit position or the 15′M2 segment Cys mutants in either subunit. We infer that propofol may bind near the extracellular end of the βsubunit M3 segment.