R-type calcium channels (RTCCs) are well known for their role in synaptic plasticity, but little is known about their subcellular distribution across various neuronal compartments. Using subtype-specific antibodies, we characterized the regional and subcellular localization of Ca(v)2.3 in mice and rats at both light and electron microscopic levels. Ca(v)2.3 immunogold particles were found to be predominantly presynaptic in the interpeduncular nucleus, but postsynaptic in other brain regions. Serial section analysis of electron microscopic images from the hippocampal CA1 revealed a higher density of immunogold particles in the dendritic shaft plasma membrane compared with the pyramidal cell somata. However, the labeling densities were not significantly different among the apical, oblique, or basal dendrites. Immunogold particles were also observed over the plasma membrane of dendritic spines, including both synaptic and extrasynaptic sites. Individual spine heads contained <20 immunogold particles, with an average density of ∼260 immunoparticles per μm(3) spine head volume, in accordance with the density of RTCCs estimated using calcium imaging (Sabatini and Svoboda, 2000). The Ca(v)2.3 density was variable among similar-sized spine heads and did not correlate with the density in the parent dendrite, implying that spines are individual calcium compartments operating autonomously from their parent dendrites.