Different subtypes of voltage‐dependent Ca2+ currents in native neurones are essential in coupling action potential firing to Ca2+ influx. For most of these currents, the underlying Ca2+ channel subunits have been identified on the basis of pharmacological and biophysical similarities. In contrast, the molecular basis of R‐type Ca2+ currents remains controversial. We have therefore examined the contribution of the CaV2.3 (α1E) subunits to R‐type currents in different types of central neurones using wild‐type mice and mice in which the CaV2.3 subunit gene was deleted. In hippocampal CA1 pyramidal cells and dentate granule neurones, as well as neocortical neurones of wild‐type mice, Ca2+ current components resistant to the combined application of ω‐conotoxin GVIA and MVIIC, ω‐agatoxin IVa and nifedipine (ICa,R) were detected that were composed of a large R‐type and a smaller T‐type component. In CaV2.3‐deficient mice, ICa,R was considerably reduced in CA1 neurones (79 %) and cortical neurones (87 %), with less reduction occurring in dentate granule neurones (47 %). Analysis of tail currents revealed that the reduction of ICa,R is due to a selective reduction of the rapidly deactivating R‐type current component in CA1 and cortical neurones. In all cell types, ICa,R was highly sensitive to Ni2+ (100 μM: 71–86 % block). A selective antagonist of cloned CaV2.3 channels, the spider toxin SNX‐482, partially inhibited ICa,R at concentrations up to 300 nm in dentate granule cells and cortical neurones (50 and 57 % block, EC50 30 and 47 nm, respectively). ICa,R in CA1 neurones was significantly less sensitive to SNX‐482 (27 % block, 300 nm SNX‐482). Taken together, our results show clearly that CaV2.3 subunits underlie a significant fraction of ICa,R in different types of central neurones. They also indicate that CaV2.3 subunits may give rise to Ca2+ currents with differing pharmacological properties in native neurones.