The GABAA receptor is a pentameric ligand-gated chloride ion channel composed of subunits from at least seven different classes (alpha1-6, beta1-3, gamma1-3, delta, epsilon, theta, pi). alpha5-containing GABAA receptors (alpha5-GABAARs) are abundantly expressed in the hippocampus, mediate a tonic conductance and are thought to be located extrasynaptically. Here we test the hypothesis that alpha5 subunit-containing GABAARs and synaptic GABAARs in hippocampal neurons are anchored differently to the plasma membrane and have distinct subunit compositions. Using surface biotinylation, biochemical fractionation and different detergent conditions, we showed that alpha5-GABAARs are predominantly expressed on the cell surface, were enriched in the soluble fraction and fail to associate with synaptic marker proteins. Double-immunofluorescence staining in hippocampal pyramidal neurons showed that alpha5-GABAARs only occasionally colocalized with synaptic marker synaptophysin, confirming its primarily extrasynaptic localization in the hippocampus. Results from co-immunoprecipitation suggested that these extrasynaptic receptors are most likely composed of the alpha5beta3gamma2 subunits.