J. Neurochem.(2012)122, 1095–1107.AbstractCa2+channel β subunits determine the maturation, biophysical properties and cell surface expression of high voltage‐activated channels. Thus, we have analysed the expression, regional distribution and subcellular localization of the Cavβ subunit family in mice from birth to adulthood. In the hippocampus and cerebellum, Cavβ1, Cavβ3and Cavβ4protein levels increased with age, although there were marked region‐ and developmental stage‐specific differences in their expression. Cavβ1was predominantly expressed in thestrata oriensandradiatumof the hippocampus, and only weakly in the cerebellum. The Cavβ3subunit was mainly expressed in thestrata radiatumandlucidumof the hippocampus and in the molecular layer of the cerebellum. During development, Cavβ3protein expression in the cerebellum peaked at postnatal days (P) 15 and 21, and had diminished drastically by P60, and in the hippocampus increased with age throughout all subfields. Cavβ4protein was detected throughout the cerebellum, particularly in the molecular layer, and in contrast to the other subunits, Cavβ4was mainly detected in the molecular layer and the hilus of the hippocampus. At the subcellular level, Cavβ1and Cavβ3were predominantly located post‐synaptically in hippocampal pyramidal cells and cerebellar Purkinje cells. Cavβ4subunits were detected in the pre‐synaptic and post‐synaptic compartments of both regions, albeit more strongly at post‐synaptic sites. These results shed new light on the developmental regulation and subcellular localization of Cavβ subunits, and their possible role in pre‐ and post‐synaptic transmission.