J. Neurochem. (2010) 114, 1805–1818. Abstract Kainate receptors (KARs) modulate neuronal network activity. The molecular mechanisms that control the assembly and trafficking of KARs are unclear. Here, we examined the role of Q/R editing and subunit composition on KAR subunit assembly and subcellular distribution. The majority of GluK2 subunits undergo editing at the Q/R site in the channel pore loop. Cell surface biotinylation, cross‐linking, Endoglycosidase‐H analysis and gradient separation of KAR subunit assembly states revealed that Q/R editing reduces oligomerization, endoplasmic reticulum (ER) export, plasma membrane expression and stability of homomeric GluK2‐containing KARs. These results indicate that Q/R editing of GluK2 may orchestrate channel subunit composition during KAR assembly in the ER. GluK2/GluK5 heteromers are the most abundant KAR subtype in the brain. While subcellular fractionation of brain tissue confirmed that both GluK2/3 and GluK5 are present in synaptosomes and tightly associated with post‐synaptic density fractions, biochemical analysis revealed that endogenous GluK2/3 subunits show less complete assembly and trafficking compared with GluK5. In transgenic mice, the loss of the key assembly partner GluK2 leads to dramatic reduction in GluK5 expression. These results support the idea that the assembly and intracellular distribution of KARs is determined by RNA editing at the Q/R site and subunit composition.