The β-subunits of voltage-gated Ca 2+ (Ca V) channels regulate the functional expression and several biophysical properties of high-voltage-activated Ca V channels. We find that Ca V β-subunits also determine channel regulation by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP 2). When Ca V1.3, -2.1, or -2.2 channels are cotransfected with the β3-subunit, a cytosolic protein, they can be inhibited by activating a voltage-sensitive lipid phosphatase to deplete PIP 2. When these channels are coexpressed with a β2a-subunit, a palmitoylated peripheral membrane protein, the inhibition is much smaller. PIP 2 sensitivity could be increased by disabling the two palmitoylation sites in the β2a-subunit. To further test effects of membrane targeting of Ca V β-subunits on PIP 2 regulation, the N terminus of Lyn was ligated onto the cytosolic β3-subunit to confer lipidation. This chimera, like the Ca V β2a-subunit, displayed plasma membrane localization, slowed the inactivation of Ca V2.2 channels, and increased the current density. In addition, the Lyn-β3 subunit significantly decreased Ca Vchannel inhibition by PIP 2 depletion. Evidently lipidation and membrane anchoring of Ca V β-subunits compete with the PIP 2 regulation of high-voltage-activated Ca V channels. Compared with expression with Ca V β3-subunits alone, inhibition of Ca V2.2 channels by PIP 2 depletion could be significantly attenuated when β2a was coexpressed with β3. Our data suggest that the Ca V currents in neurons would be regulated by membrane PIP 2 to a degree that depends on their endogenous β-subunit combinations.