The beta-subunit is an integral component of purified voltage-sensitive Ca2+ channels. 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. 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. 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 functionally 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.