The multisubunit (α_1S, α₂/δ, β₁, and γ) skeletal muscle dihydropyridine receptor transduces transverse tubule membrane depolarization into release of Ca²⁺ from the sarcoplasmic reticulum, and also acts as an L-type Ca²⁺ channel. The α_1S subunit contains the voltage sensor and channel pore, the kinetics of which are modified by the other subunits. To determine the role of the β₁ subunit in channel activity and excitation-contraction coupling we have used gene targeting to inactivate the β₁ gene. β₁-null mice die at birth from asphyxia. Electrical stimulation of β₁-null muscle fails to induce twitches, however, contractures are induced by caffeine. In isolated β₁-null myotubes, action potentials are normal, but fail to elicit a Ca²⁺ transient. L-type Ca²⁺ current is decreased 10- to 20-fold in the β₁-null cells compared with littermate controls. Immunohistochemistry of cultured myotubes shows that not only is the β₁ subunit absent, but the amount of α_1S in the membrane also is undetectable. In contrast, the β₁ subunit is localized appropriately in dysgenic, mdg/mdg, (α_1S-null) cells. Therefore, the β₁ subunit may not only play an important role in the transport/insertion of the α_1S subunit into the membrane, but may be vital for the targeting of the muscle dihydropyridine receptor complex to the transverse tubule/sarcoplasmic reticulum junction.