To study the interactions of the alpha1S subunit of the skeletal muscle L-type Ca2+ channel with the skeletal beta1a and the cardiac beta2a, these subunits were expressed alone or in combination in tsA201 cells. Immunofluorescence- and green fluorescent protein-labeling showed that, when expressed alone, beta1a was diffusely distributed throughout the cytoplasm, beta2a was localized in the plasma membrane, and alpha1S was concentrated in a tubular/reticular membrane system, presumably the endoplasmic reticulum (ER). Upon coexpression with alpha1S, beta1a became colocalized with alpha1S in the ER. Upon coexpression with beta2a, alpha1S redistributed to the plasma membrane, where it aggregated in large clusters. Coexpression of alpha1S with beta1a but not with beta2a increased the frequency at which cells expressed L-type currents. A point mutation (alpha1S-Y366S) or deletion (alpha1S-Delta351-380) in the beta interaction domain of alpha1S blocked both translocation of beta1a to the ER and beta2a-induced translocation of the alpha1S mutants to the plasma membrane. However, the point mutation did not interfere with beta1a-induced current stimulation. Thus, beta1a and beta2a are differentially distributed in tsA201 cells and upon coexpression with alpha1S, form alpha1S. beta complexes in different cellular compartments. Complex formation but not current stimulation requires the intact beta interaction domain in the I-II cytoplasmic loop of alpha1S.