R-type Ca²⁺ channels cooperate with P/Q- and N-type channels to control neurotransmitter release at central synapses. The leading candidate as pore-forming subunit of R-type channels is the α_1E subunit. However, R-type Ca²⁺ currents with permeation and/or pharmacological properties different from those of recombinant Ca²⁺channels containing α_1E subunits have been described, and therefore the molecular nature of R-type Ca²⁺channels remains not completely settled. Here, we show that the R-type Ca²⁺ current of rat cerebellar granule cells consists of two components inhibited with different affinity by the α_1E selective antagonist SNX482 (IC₅₀ values of 6 and 81 nm) and a third component resistant to SNX482. The SNX482-sensitive R-type current shows the unique permeation properties of recombinant α_1E channels; it is larger with Ca²⁺ than with Ba²⁺ as charge carrier, and it is highly sensitive to Ni²⁺ block and has a voltage-dependence of activation consistent with that of G2 channels with unitary conductance of 15 pS. On the other hand, the SNX482-resistant R-type current shows permeation properties similar to those of recombinant α_1A and α_1B channels; it is larger with Ba²⁺ than with Ca²⁺ as charge carrier_, and it has a low sensitivity to Ni²⁺ block and a voltage-dependence of activation consistent with that of G3 channels with unitary conductance of 20 pS. Gene-specific knock-down by antisense oligonucleotides demonstrates that the different cerebellar R-type channels are all encoded by the α_1E gene, suggesting the existence of α_1E isoforms with different pore properties.