We have cloned two splice variants of the human homolog of the alpha1A subunit of voltage-gated Ca2+ channels. The sequences of human alpha1A-1 and alpha1A-2 code for proteins of 2510 and 2662 amino acids, respectively. Human alpha1A-2alpha2bdeltabeta1b Ca2+ channels expressed in HEK293 cells activate rapidly (tau+10mV = 2.2 ms), deactivate rapidly (tau-90mV = 148 micros), inactivate slowly (tau+10mV = 690 ms), and have peak currents at a potential of +10 mV with 15 mM Ba2+ as charge carrier. In HEK293 cells transient expression of Ca2+ channels containing alpha1A/B(f), an alpha1A subunit containing a 112 amino acid segment of alpha1B-1 sequence in the IVS3-IVSS1 region, resulted in Ba2+ currents that were 30-fold larger compared to wild-type (wt) alpha1A-2-containing Ca2+ channels, and had inactivation kinetics similar to those of alpha1B-1-containing Ca2+ channels. Cells transiently transfected with alpha1A/B(f)alpha2bdeltabeta1b expressed higher levels of the alpha1, alpha2bdelta, and beta1b subunit polypeptides as detected by immunoblot analysis. By mutation analysis we identified two locations in domain IV within the extracellular loops S3-S4 (N1655P1656) and S5-SS1 (E1740) that influence the biophysical properties of alpha1A. alpha1AE1740R resulted in a threefold increase in current magnitude, a -10 mV shift in steady-state inactivation, and an altered Ba2+ current inactivation, but did not affect ion selectivity. The deletion mutant alpha1ADeltaNP shifted steady-state inactivation by -20 mV and increased the fast component of current inactivation twofold. The potency and rate of block by omega-Aga IVA was increased with alpha1ADeltaNP. These results demonstrate that the IVS3-S4 and IVS5-SS1 linkers play an essential role in determining multiple biophysical and pharmacological properties of alpha1A-containing Ca2+ channels.