The skeletal muscle L-type Ca2+ channel is a complex of five subunits that is specifically localized in the triad. Its primary function is the rapid activation of Ca2+ release from cytoplasmic stores in a process called excitation-contraction coupling. To study the role of α1S–β1a interactions in the incorporation of the functional channel complex into the triad, α1S and β1a [or a β1a-green fluorescent protein (GFP) fusion protein] were expressed alone and in combination in myotubes of the dysgenic cell line GLT. βGFP expressed in dysgenic myotubes that lack the skeletal muscle α1S subunit was diffusely distributed in the cytoplasm. On coexpression with the α1S subunit βGFP distribution became clustered and colocalized with α1S immunofluorescence. Based on the colocalization of βGFP and α1S with the ryanodine receptor the clusters were identified as T-tubule/sarcoplasmic reticulum junctions. Expression of α1S with and without β1a restored Ca2+ currents and depolarization-induced Ca2+ release. The translocation of βGFP from the cytoplasm into the junctions failed when βGFP was coexpressed with α1S mutants in which the β interaction domain had been altered (α1S-Y366S) or deleted (α1S-Δ351–380). Although α1S-Y366S did not associate with βGFP it was incorporated into the junctions, and it restored Ca2+ currents and depolarization-induced Ca2+ release. Thus, β1a requires the association with the β interaction domain in the I–II cytoplasmic loop of α1S for its own incorporation into triad junctions, but stable α1S–β1a association is not necessary for the targeting of α1S into the triads or for its normal function in Ca2+ conductance and excitation-contraction coupling.