Voltage-gated calcium channels (Cav) mediate calcium influx in living cells and are necessary for essential physiological functions such as muscle contraction and excitation-transcription coupling. Channels of the Cav2 family (Cav2.1, 2.2, and 2.3) are highly expressed in the central nervous system (CNS) and are important regulators of neuronal excitability. They are involved in pre- and postsynaptic functions where they, for example, trigger neurotransmitter release. Cav2.3 (α1E) is involved in neuronal development and, when compared to other α1 subunits, shows the strongest expression in mouse hippocampus. Nevertheless, little is known about the subcellular localization of Cav2.3 in CNS neurons. Here, we aim to investigate the pre- and postsynaptic localization of Cav2.3 in hippocampal neurons. To this end, we are employing low-density primary cultures of mouse hippocampal neurons, transfected with HA-epitope tagged α1 subunits of calcium channels, immunofluorescence staining, and high-resolution fluorescence microscopy. For a comparative quantitative analyses, the well-characterized Cav1.2 (α1C) channel is used as control. Analysis of live cell labelled hippocampal neurons revealed a clustered localization of Cav2.3 channels throughout the neuronal plasma membrane of somata, dendrites, and axons. In addition, Cav2.3 channels show a strong presynaptic expression in excitatory (glutamatergic) and inhibitory (GABAergic) neurons. However, and similar to Cav1.2, Cav2.3 also shows a strong expression in dendritic spines of excitatory glutamatergic synapses and in GABAergic postsynaptic locations opposite vGAT positive synaptic boutons. Taken together, our results show a pre- and postsynaptic localization pattern of Cav2.3 channels, which supports its proposed roles in synaptic transmission and postsynaptic calcium signalling.