Transmembrane AMPA receptor regulatory proteins (TARPs) are AMPA receptor auxiliary subunits that influence diverse aspects of receptor function. However, the full complement of physiological roles for TARPs in vivo remains poorly understood. Here we find that double knock-out mice lacking TARPs gamma-2 and gamma-3 are profoundly ataxic and fail to thrive. We demonstrate that these TARPs are critical for the synaptic targeting and kinetics of AMPA receptors in cerebellar Golgi cells, but that either alone is sufficient to fully preserve function. By analyzing the few remaining synaptic AMPA receptors in the gamma-2, gamma-3 double knock-out mice, we unexpectedly find that these TARPs specify AMPA receptor subunit composition. This study establishes a new role for TARPs in regulating AMPA receptor assembly and suggests that TARPs are necessary for proper AMPA receptor localization and function in most, if not all, neurons of the CNS.