The accessory α₂δ subunits of voltage-gated calcium channels are highly glycosylated transmembrane proteins that interact with calcium channel α1 subunits to enhance calcium currents. We compared the membrane localization and processing of native cerebellar α₂δ-2 subunits with α₂δ-2 stably expressed in tsA-201 cells. We identified that α₂δ-2 is completely concentrated in cholesterol-rich microdomains (lipid rafts) in cerebellum, in which it substantially colocalizes with the calcium channel α1 subunit Ca_V2.1, although Ca_V2.1 is also present in the Triton X-100-soluble fraction. In tsA-201 cells, unlike cerebellum, α₂δ-2 is not completely proteolytically processed into α₂-2 and δ-2. However, this processing is more complete in the lipid raft fraction of tsA-201 cells, in which α₂δ-2 also colocalizes with Ca_V2.1. Cholesterol depletion of intact cells disrupted their lipid rafts and enhanced Ca_V2.1/α₂δ-2/β4 currents. Furthermore, α₂δ-2 coimmunoprecipitates with lipid raft-associated proteins of the stomatin family. The apparent affinity of α₂δ-2 for its ligand gabapentin is increased markedly in the cholesterol-rich microdomain fractions, in both cerebellum and the stable α₂δ-2 cell line. In contrast, α₂δ-2 containing a point mutation (R282A) has a much lower affinity for gabapentin, and this is not enhanced in the lipid raft fraction. This R282A mutant α₂δ-2 shows reduced functionality in terms of enhancement of Ca_V2.1/β4 calcium currents, suggesting that the integrity of the gabapentin binding site may be important for normal functioning of α₂δ-2. Together, these results indicate that both α₂δ-2 and Ca_V2.1 are normally associated with cholesterol-rich microdomains, and this influences their functionality.