Increased plasmalemmal localization of α4βδ GABA(A) receptors (GABARs) occurs in the hippocampal pyramidal cells of female mice at pubertal onset (Shen et al., 2010). This increase occurs on both dendritic spines and shafts of CA1 pyramidal cells and is in response to hormone fluctuations that occur at pubertal onset. However, little is known about how the α4 and δ subunits individually mediate the formation of functional, plasmalemmal α4βδ GABARs. To determine whether expression of the α4 subunit is necessary for plasmalemmal δ subunit localization at pubertal onset, electron microscopic-immunocytochemistry (EM-ICC) was employed. CA1 pyramidal cells of female α4 knockout (KO) mice were tested for plasmalemmal levels of the δ subunit within dendritic spine and shaft profiles at the onset of puberty. EM-ICC revealed that the α4 and δ subunits localize on dendritic spines and shafts at sites extrasynaptic to GABAergic input at pubertal onset in tissue of wild-type (WT) mice. At pubertal onset, plasmalemmal localization of the δ subunit is reduced 45.9% on dendritic spines, and 56.7% on dendritic shafts with KO of the α4 subunit, as compared to WT tissue, yet levels of intracellular δ immunoreactivity remain unchanged. The decline in plasmalemmal localization is manifested as decreased responsiveness to the GABA agonist gaboxadol at concentrations that are selective for δ-containing GABARs. Additionally, α4 KO mice have larger dendritic spine and shaft profiles. Our findings demonstrate that α4 subunit expression strongly influences the pubertal increase of δ subunits at the plasma membrane, and that genetic deletion of α4 serves as a functional knock-down of δ-containing GABARs.