The auxiliary β subunit is essential for functional expression of high voltage-activated Ca²⁺ channels. This effect is partly mediated by a facilitation of the intracellular trafficking of α₁ subunit toward the plasma membrane. Here, we demonstrate that the I-II loop of the α₁ subunit contains an endoplasmic reticulum (ER) retention signal that severely restricts the plasma membrane incorporation of α₁ subunit. Coimmunolabeling reveals that the I-II loop restricts expression of a chimera CD8-I-II protein to the ER. The β subunit reverses the inhibition imposed by the retention signal. Extensive deletion of this retention signal in full-length α₁ subunit facilitates the cell surface expression of the channel in the absence of β subunit. Our data suggest that the β subunit favors Ca²⁺ channel plasma membrane expression by inhibiting an expression brake contained in β-binding α₁ sequences.