Two C-terminal splice variants (BI-1 and BI-2, now termed Cav2.1a and Cav2.1b) of the neuronal voltage-gated P/Q-type Ca2+ channel α1A pore-forming subunit have been cloned (Mori et al., 1991, Nature, 350, 398–402). BI-1 and BI-2 code for proteins of 2273 and 2424 amino acids, respectively, and differ only by their extreme carboxyl-termini sequences. Here, we show that, in Xenopus oocytes, the two isoforms direct the expression of channels with different properties. Electrophysiological analysis showed that BI-1 and BI-2 have peak Ba2+ currents (IBa) at a potential of +30 and +20 mV, respectively. The different C-terminal sequence (amino acids 2229–2273) of BI-1 caused a shift in steady-state inactivation by +10 mV and decreased the proportion of fast component of current inactivation twofold. Likewise, the biophysical changes in IBa caused by coexpression of the β4 auxiliary subunit were substantially different in BI-1- and BI-2-containing channels in comparison to those induced by β3. Several of these differences in β regulation were abolished by deleting the carboxyl-terminal splicing region. By creating a series of GST fusion proteins, we identified two locations in the C-terminal (Leu2090–Gly2229 for BI-1 and BI-2, and Arg2230–Pro2424 for BI-2 only) that determine the differential interaction of β4 with the distinct α1A isoforms. These interactions appear to favour the binding of β4 to the AID site, and also the plasma membrane expression of BI-2. These results demonstrate that the final segment of the C-terminal affects α1A channel gating, interaction and regulation with/by the β subunits. The data will have several implications for the understanding of the biophysical effects of many channelopathies in which the carboxyl-termini of α1A and β4 are affected.