Pregabalin, a synthetic branched chain γ-amino acid with anticonvulsant, anxiolytic, and analgesic activities, has been shown to bind with high affinity to the voltage-gated calcium channel α(2)δ subunit. Given the broad therapeutic utility of pregabalin, a series of experiments was undertaken to determine the potency, selectivity, and specificity of pregabalin's receptor-binding profile at α(2)δ-1 and α(2)δ-2 subunits of voltage-gated calcium channels along with 38 widely studied receptors and channels. Receptor autoradiography was used to assess regional-binding density of pregabalin throughout the rat spinal cord and brain. In addition, a series of studies using in vivo electrophysiological recordings of γ-aminobutyric acid (GABA)(A)- and GABA(B)-evoked currents was undertaken to determine the interaction of pregabalin with GABAergic receptor subtypes. Together, the results of these studies demonstrate potent and selective binding of pregabalin to α(2)δ-1 and α(2)δ-2 subunits in native and recombinant human and porcine systems. Pregabalin did not interact with any of the 38 receptors and ion channels evaluated, and a variety of central nervous system (CNS)-targeted therapeutic drugs did not show activity at the α(2)δ subunits of voltage-gated calcium channels. Receptor autoradiography demonstrated extensive [(3)H]-pregabalin binding throughout the CNS, with high-level binding in the cortex, hippocampus, cerebellum, dorsal horn of the spinal cord, and amygdala. Finally, receptor-binding and electrophysiological techniques failed to show evidence of an interaction between pregabalin and GABA(A) or GABA(B) receptors. These studies suggest that the clinical effects of pregabalin are likely due to direct and selective interactions with α(2)δ-1 and α(2)δ-2 subunits of voltage-gated calcium channels.