A fundamental objective of anesthesia research is to identify the receptors and brain regions that mediate the various behavioral components of the anesthetic state, including amnesia, immobility, and unconsciousness. Using complementary in vivo and in vitro approaches, we found that GABA_A receptors that contain the α5 subunit (α5GABA_ARs) play a critical role in amnesia caused by the prototypic intravenous anesthetic etomidate. Whole-cell recordings from hippocampal pyramidal neurons showed that etomidate markedly increased a tonic inhibitory conductance generated by α5GABA_ARs, whereas synaptic transmission was only slightly enhanced. Long-term potentiation (LTP) of field EPSPs recorded in CA1 stratum radiatum was reduced by etomidate in wild-type (WT) but not α5 null mutant (α5−/−) mice. In addition, etomidate impaired memory performance of WT but not α5−/− mice for spatial and nonspatial hippocampal-dependent learning tasks. The brain concentration of etomidate associated with memory impairment in vivo was comparable with that which increased the tonic inhibitory conductance and blocked LTP in vitro. The α5−/− mice did not exhibit a generalized resistance to etomidate, in that the sedative-hypnotic effects measured with the rotarod, loss of righting reflex, and spontaneous motor activity were similar in WT and α5−/− mice. Deletion of the α5 subunit of the GABA_ARs reduced the amnestic but not the sedative-hypnotic properties of etomidate. Thus, the amnestic and sedative-hypnotic properties of etomidate can be dissociated on the basis of GABA_AR subtype pharmacology.