The γ-aminobutyric acid type A (GABAA) receptor is a pentameric ligand-gated ion channel responsible for fast synaptic inhibition in the brain. Phosphorylation of the GABAA receptor by serine/threonine protein kinases, at residues located in the intracellular loop between the third and fourth transmembrane domains of each subunit, can dynamically modulate receptor trafficking and function. In this study, we have assessed the effect that Ca2+-calmodulin-dependent protein kinase-II (CaMK-II) has on GABAA receptors. The intracellular application of preactivated CaMK-II failed to modulate the function of αβ and αβγ subunit GABAA receptors heterologously expressed in human embryonic kidney (HEK)293 cells. However, application of similarly preactivated α-CaMK-II significantly potentiated the amplitudes of whole-cell GABA currents recorded from rat cultured cerebellar granule neurons and from recombinant GABAA receptors expressed in neuroblastoma, NG108-15, cells. The modulation by α-CaMK-II of current amplitude depended upon the subunit composition of GABAA receptors. α-CaMK-II potentiated GABA currents recorded from α1β3 and α1β3γ2 GABAA receptors, but was unable to functionally modulate β2 subunit-containing receptors. Similar results were obtained from β2 –/– mouse cerebellar granule cell cultures and from rat granule cell cultures overexpressing recombinant α1β2 or α1β3 GABAA receptors. α-CaMK-II had a greater effect on the modulation of GABA responses mediated by α1β3γ2 compared with α1β3 receptors, indicating a possible role for the γ2 subunit in CaMK-II-mediated phosphorylation. In conclusion, CaMK-II can upregulate the function of GABAA receptors expressed in neurons or a neuronal cell line that is dependent on the β subunit co-assembled into the receptor complex.