Benzodiazepine binding to gamma-aminobutyric acid type A (GABA(A)) receptors allosterically modulates GABA binding and increases the currents induced by submaximal GABA concentrations. Benzodiazepines induce conformational changes in the GABA-binding site in the extracellular domain, but it is uncertain whether these conformational changes extend into the membrane-spanning domain where the channel gate is located. Alone, benzodiazepines do not open the channel. We used the substituted-cysteine-accessibility method to investigate diazepam-induced conformational changes in the region of the alpha(1)-subunit M3 membrane-spanning segment. In the absence of diazepam or GABA, pCMBS(-) did not react at a measurable rate with cysteine-substitution mutants between alpha(1)Phe296 and alpha(1)Glu303. In the presence of 100 nM diazepam, pCMBS(-) reacted with alpha(1)F296C, alpha(1)F298C, and alpha(1)L301C but not with the other cysteine mutants between alpha(1)Phe296 and alpha(1)Glu303. These three mutants are a subset of the five residues that we previously showed reacted with pCMBS(-) applied in the presence of GABA. The pCMBS(-) reaction rates with these three cysteine mutants were similar in the presence of diazepam and GABA. Thus, diazepam, which binds to the extracellular domain, induces a conformational change in the membrane-spanning domain that is similar to a portion of the change induced by GABA. Because diazepam does not open the channel, these results provide structural evidence that the diazepam-bound state represents an intermediate conformation distinct from the open and resting/closed states of the receptor. The diazepam-induced conformational change in the M3 segment vicinity may be related to the mechanism of allosteric potentiation.