Three ionotropic glutamate receptor subunits, designated GluRIIA, GluRIIB, and GluRIII, have been identified at neuromuscular junctions ofDrosophila. Whereas GluRIIA and GluRIIB are redundant for viability, it was shown recently that GluRIII is essential for both the synaptic localization of GluRIIA and GluRIIB and the viability ofDrosophila. Here we identify a fourth and a fifth subunit expressed in the neuromuscular system, which we name GluRIID and GluRIIE. Both new subunits we show to be necessary for survival. Moreover, both GluRIID and GluRIIE are required for the synaptic expression of all other glutamate receptor subunits. All five subunits are interdependent for receptor function, synaptic receptor expression, and viability. This indicates that synaptic glutamate receptors incorporate the GluRIII, GluRIID, and GluRIIE subunit together with either GluRIIA or GluRIIB at theDrosophilaneuromuscular junction. At this widely used model synapse, the assembly of four different subunits to form an individual glutamate receptor channel may thus be obligatory. This study opens the way for a further characterization ofin vivoglutamate receptor assembly and trafficking using the efficient genetics ofDrosophila.