THE β-subunit is an integral component of purified voltage-sensitive Ca²⁺ channels^1–3. Modulation of Ca²⁺ channel activity by the β-subunit, which includes significant increases in transmembrane current and/or changes in kinetics, is observed on coexpression of six α₁-subunit genes with four β-subunit genes in all α₁- β combinations tested^4–12. Recent reports suggest that this regulation is not due to targeting of the α₁-subunit to the plasma membrane but is probably a result of a conformational change induced by the β-subunit^11,13. Here we report that the β-subunit binds to the cytoplasmic linker between repeats I and II of the dihydropyridine-sensitive α₁-subunits from skeletal (α_1S) and cardiac muscles (α_1C-a), and also with the more distantly related neuronal α_1A and ω-conotoxin GVIA-sensitive aα_1B-subunits. Sequence analysis of the β-subunit binding site identifies a conserved motif (QQ-E--L-GY--WI---E) positioned 24 amino acids from the IS6 transmem-brane domain in each α₁-subunit. Mutations within this motif reduce the stimulation of peak currents by the β-subunit and alter inactivation kinetics and voltage-dependence of activation. Conser-vation of the β-subunit binding motif in these functionally distinct calcium channels suggests a critical role for the I-II cytoplasmic linker of the α₁-subunit in channel modulation by the β-subunit.