The rat brain α1A calcium channel clone has been expressed in COS-7 cells together with the neuronal accessory subunits p1b and α2-δ. From reverse transcriptase polymerase chain reaction (RT-PCR), immunocytochemistry and electrophysiology experiments, we have obtained no evidence that these cells contain any endogenous calcium channels. Transfected cells were identified by co-expression of a cDNA for the reporter Green Fluorescent Protein. From immunocytochemical evidence, a high degree of co-expression was obtained between Green Fluorescent Protein and individual calcium channel subunits. When all three calcium channel subunits (α1, α2-δ and β1b) were co-expressed, evidence was obtained that all subunits were present at the cell membrane. Voltage-dependent calcium currents were observed between 24 and 72 h after transfection with the three calcium channel subunits. The current density for the combination α1A/α/β1ba24IPlb was 4.19 ± 0.69 pA.pF-1 and the current produced was slowly inactivating. The time constant of inactivation of the maximum IBa was 332 ± 46 ms (n = 5). The voltage-dependence of activation and steady-state inactivation had voltages of half activation and inactivation of 9.5 ± 2.5 mV and -30.4 ± 1.5 mV respectively, and there was little overlap between the two curves. The α1A current was completely blocked by 100 μM Cd2+ and was also blocked by ω-conotoxin MVIIC (500 nM). Dose-inhibition curves and analysis of kon and kfor for ω-agatoxin IVA both revealed apparent KD values of approximately 11 nM for α1A currents, with a kD of 7.8 × 104 M-1s-1. The results suggest that α1A expressed in these cells has some resemblance to the P type component of calcium current observed in native neurons, although it shows a somewhat greater degree of inactivation than native P current, more similar to the Q type current component. It also has an affinity for ω-agatoxin IVA 2–5 fold lower than reported for P current, but approximately 9-fold higher than reported for Q current.