We have investigated the contribution of the Cavβ subunits to the process of inactivation dependent of the I–II loop of Cavα2.1. Two amino acid residues located in the alpha1 interaction domain (AID) of the I–II loop of Cavα2.1 (Arg387 and Glu388) have been directly implicated in voltage-dependent inactivation of this channel. Various point mutations of these residues disrupt the interaction between the I–II loop and the III–IV loop, and thereby modify the inactivation properties of the channel by accelerating its kinetics and shifting the steady-state inactivation curve towards hyperpolarized potentials. A similar disruption is produced by Cavβ4 subunit association with the I–II loop. Moreover, in the presence of Cavβ4 subunit, introducing negatively charged residues at positions 387 or 388 slows inactivation kinetics down, whereas introducing positive charges has the opposite effect. The shift of the steady-state inactivation curve is also amino acid charge-dependent. In contrast, mutation of Arg387 or Glu388 does not alter the differential regulation of the different Cavβ isoforms on inactivation. These results suggest that the expression of Cavβ4 alters the contribution of charged residues at positions 387 and 388 to inactivation. We discuss these results with regard to the actual hypotheses on the mechanisms of calcium channel inactivation. We introduce the working concept that Cavβ-subunits produce a conformational repositioning of charged AID residues within the electric field.