High-voltage-activated Ca2+channels are essential for diverse biological processes. They are composed of four or five subunits, including α1, α2-δ, β and γ (ref.1). Their expression and function are critically dependent on the β-subunit, which transports α1to the surface membrane and regulates diverse channel properties2-4. It is believed3-6that the β-subunit interacts with α1primarily through the β-interaction domain (BID), which binds directly to the α-interaction domain (AID) of α17; however, the molecular mechanism of the α1-β interaction is largely unclear. Here we report the crystal structures of the conserved core region of β3, alone and in complex with AID, and of β4alone. The structures show that the β-subunit core contains two interacting domains: a Src homology 3 (SH3) domain and a guanylate kinase (GK) domain. The AID binds to a hydrophobic groove in the GK domain through extensive interactions, conferring extremely high affinity between α1and β-subunits4,8. The BID is essential both for the structural integrity of and for bridging the SH3 and GK domains, but it does not participate directly in binding α1. The presence of multiple protein-interacting modules in the β-subunit opens a new dimension to its function as a multi-functional protein.