Elevation of intracellular pH (pH_i) enhances the activity of native L‐type Ca²⁺ channels in cardiac and smooth muscle. We studied the modulation by pH_i of expressed L‐type Ca²⁺ channels comprised of either the α_1C subunits alone or of α_1C plus β_2a subunits. Ca²⁺ channels were expressed in human embryonic kidney cells (HEK 293) and pH_i was increased from a basal level of 7.3 to 8.3 by exposure of cells to NH₄Cl (20 mM) or by elevation of extracellular pH to 8.5. Elevation of pH_i enhanced the activity of Ca²⁺ channels derived by coexpression of α_1C and β_2a subunits. This alkalosis‐induced stimulation of channel activity was mainly due to an increase in channel availability. Channels derived by expression of α_1C alone were not affected by intracellular alkalosis. Our results demonstrate that the pH_i sensitivity of L‐type Ca²⁺ channels is conferred by the β subunit of the channel complex.