Voltage-dependent calcium channels selectively enable Ca2+ ion movement through cellular membranes. These multiprotein complexes are involved in a wide spectrum of biological processes such as signal transduction and cellular homeostasis. α1 is the membrane pore-forming subunit, whereas β is an intracellular subunit that binds to α1, facilitating and modulating channel function. We have expressed, purified, and characterized recombinant β3 and β2a using both biochemical and biophysical methods, including electrophysiology, to better understand the β family's protein structural and functional correlates. Our results indicate that the β protein is composed of two distinct domains that associate with one another in a stable manner. The data also suggest that the polypeptide regions outside these domains are not structured when β is not in complex with the channel. In addition, the β structural core, comprised of just these two domains without other sequences, binds tightly to the α interaction domain (AID) motif, a sequence derived from the α1 subunit and the principal anchor site of β. Domain II is responsible for this binding, but domain I enhances it.