Voltage-gated calcium channels (VGCCs) play an essential role in controlling neurotransmitter release, neuronal excitability, and gene expression in the nervous system. The distribution of cells that contain mRNAs encoding the auxiliary α2δ-1, α2δ-2, and α2δ-3 subunits of the VGCCs in the central nervous system (CNS) and the dorsal root ganglia (DRG) was examined in rats by using in situ hybridization. Specific labeling of α2δ-1, α2δ-2, and α2δ-3 mRNAs appeared to be largely confined to neurons and was widely, although differentially, distributed in the brain, the spinal cord, and the DRG. Importantly, α2δ-2 mRNA was found to be expressed in interneurons in the cortex, the hippocampus, the striatum, and in regions that contain dense cholinergic neurons. Our results suggest that different α2δ subunits may exert distinctive functions in the CNS. The α2δ-1 subunit mRNA is localized in brain regions known to be involved in cortical processing, learning and memory, defensive behavior, neuroendocrine secretion, autonomic activation, primary sensory transmission, and general arousal. The α2δ-2 subunit mRNA is present in brain regions known to modulate the overall activities of the cortex, the hippocampus, and the thalamus. The α2δ-2 subunit is also found in brain regions known to be involved in olfaction, somatic motor control, fluid homeostasis, ingestive and defensive behaviors, neuroendocrine functions, and circadian rhythm. In addition to being localized in brain regions that express α2δ-1 and α2δ-2 subunit mRNAs, α2δ-3 subunit mRNA is highly expressed in regions involved in auditory information processing and somatic movement. J. Comp. Neurol. 491:246–269, 2005. © 2005 Wiley-Liss, Inc.