The effects of auxiliary calcium channel subunits on the expression and functional properties of high-voltage activated (HVA) calcium channels have been studied extensively in the Xenopus oocyte expression system, but are less completely characterized in a mammalian cellular environment. Here, we provide the first systematic analysis of the effects of calcium channel β and α2–δ subunits on expression levels and biophysical properties of three different types (Cav1.2, Cav2.1 and Cav2.3) of HVA calcium channels expressed in tsA-201 cells. Our data show that Cav1.2 and Cav2.3 channels yield significant barium current in the absence of any auxiliary subunits. Although calcium channel β subunits were in principle capable of increasing whole cell conductance, this effect was dependent on the type of calcium channel α1 subunit, and β3 subunits altogether failed to enhance current amplitude irrespective of channel subtype. Moreover, the α2–δ subunit alone is capable of increasing current amplitude of each channel type examined, and at least for members of the Cav2 channel family, appears to act synergistically with β subunits. In general agreement with previous studies, channel activation and inactivation gating was regulated both by β and by α2–δ subunits. However, whereas pronounced regulation of inactivation characteristics was seen with the majority of the auxiliary subunits, effects on voltage dependence of activation were only small (< 5 mV). Overall, through a systematic approach, we have elucidated a previously underestimated role of the α2–δ1 subunit with regard to current enhancement and kinetics. Moreover, the effects of each auxiliary subunit on whole cell conductance and channel gating appear to be specifically tailored to subsets of calcium channel subtypes.