Surface expression level of voltage-dependent calcium channels is tightly controlled in neurons to avoid the resulting cell toxicity generally associated with excessive calcium entry. Cell surface expression of high voltage-activated calcium channels requires the association of the pore-forming subunit, Cavalpha, with the auxiliary subunit, Cavbeta. In the absence of this auxiliary subunit, Cavalpha is retained in the endoplasmic reticulum (ER) through mechanisms that are still poorly understood. Here, we have investigated, by a quantitative method based on the use of CD8 alpha chimeras, the molecular determinants of Cavalpha2.1 that are responsible for the retention, in the absence of auxiliary subunits, of P/Q calcium channels in the ER (referred to here as 'ER retention'). This study demonstrates that the I-II loop of Cavalpha2.1 contains multiple ER-retention determinants beside the beta subunit association domain. In addition, the I-II loop is not the sole domain of calcium channel retention as two regions identified for their ability to interact with the I-II loop, the N- and C-termini of Cavalpha2.1, also produce ER retention. It is also not an obligatory determinant as, similarly to low-threshold calcium channels, the I-II loop of Cavalpha1.1 does not produce ER retention in COS7 cells. The data presented here suggests that ER retention is suppressed by sequential molecular events that include: (i). a correct folding of Cavalpha in order to mask several internal ER-retention determinants and (ii). the association of other proteins, including the Cavbeta subunit, to suppress the remaining ER-retention determinants.