To study the interactions of the alpha(1S) subunit of the skeletal muscle L-type Ca2+ channel with the skeletal beta(1a) and the cardiac beta(2a), these subunits were expressed alone or in combination in tsA201 cells, Immunofluo rescence- and green fluorescent protein-labeling showed that, when expressed alone, beta(1a) was diffusely distributed throughout the cytoplasm, beta(2a) was localized in the plasma membrane, and alpha(1S) was concentrated in a tubular/reticular membrane system, presumably the endoplasmic reticulum (ER), Upon coexpression with alpha(1S), beta(1a) became colocalized with alpha(1S) in the ER, Upon coexpression with beta(2a), alpha(1S) redistributed to the plasma membrane, where it aggregated in large clusters, Coexpression of alpha(1S) with beta(1a) but not with beta(2a) increased the frequency at which cells expressed L-type currents, A point mutation (alpha(1S)-Y366S) or deletion (alpha(1S)-Delta 351-380) in the beta interaction domain of alpha(1S) blocked both translocation of beta(1a) to the ER and beta(2a)-induced translocation of the alpha(1S) mutants to the plasma membrane, However, the point mutation did not interfere with beta(1a)-induced current stimulation, Thus, beta(1a) and beta(2a) are differentially distributed in tsA201 cells and upon coexpression with alpha(1S), form alpha(1S).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 alpha(1S).