THE beta-subunit is an integral component of purified voltage-sensitive Ca2+ channels(1-3). Modulation of Ca2+ channel activity by the beta-subunit, which includes significant increases in transmembrane current and/or changes in kinetics, is observed on coexpression of six alpha(1)-subunit genes with four beta-subunit genes in all alpha(1)-beta combinations tested(4-12). Recent reports suggest that this regulation is not due to targeting of the alpha(1)-subunit to the plasma membrane but is probably a result of a conformational change induced by the beta-subunit(11,13). Here we report that the beta-subunit binds to the cytoplasmic linker between repeats I and II of the dihydropyridine-sensitive alpha(1)-subunits from skeletal (alpha(1S)) and cardiac muscles (alpha(1C-a)), and also with the more distantly related neuronal alpha(1A) and omega-conotoxin GVIA-sensitive alpha 1B-subunits. Sequence analysis of the beta-subunit binding site identifies a conserved motif (QQ-E--L-GY--WI---E) positioned 24 amino acids from the IS6 transmembrane domain in each alpha(1)-subunit. Mutations within this motif reduce the stimulation of peak currents by the beta-subunit and alter inactivation kinetics and voltage-dependence of activation. Conservation of the beta-subunit binding motif in these functonally distinct calcium channels suggests a critical role for the I-II cytoplasmic linker of the alpha(1)-subunit in channel modulation by the beta-subunit.