G protein-gated inwardly rectifying potassium (Kir3) channels are involved in regulating membrane excitability in the brain. Kir3 channels have been shown to play a role in learning, analgesia and drug addiction. Little is known about the cell surface regulation of Kir3 channels. Using a proteomics approach, we recently discovered that sorting nexin 27 (SNX27) associates with a subset of Kir3 channels. Sorting nexins have been implicated in trafficking of proteins through endosomal compartments. The single PDZ domain of SNX27 binds directly to the PDZ binding motif of Kir3 channels leading to their downregulation. Here, we examined the functional effect of SNX27b expression on different subunit combinations of the Kir3 family. Our results show that regulation of Kir3 channels by SNX27 depends critically on the combination of Kir3 subunits. This type of subunit-specific regulation could be important for determining the extent of Kir3 inhibition in normal as well as diseased states, such as drug addiction.