Although pharmacological and electrophysiological studies have demonstrated the existence of multiple types of voltage-dependent calcium channels in neuronal tissue, the subunit composition of these channels is not well known. Here, we report the cloning and expression of a new rat brain beta subunit (beta 4). Northern blot analysis indicates that beta 4 mRNA is expressed almost exclusively in neuronal tissues, with the highest levels being found in the cerebellum. Coexpression studies indicate that rat beta 4 can interact with rabbit cardiac muscle alpha 1, rabbit skeletal muscle alpha 1, and calcium channels endogenous to Xenopus oocytes. beta 4 modulation of alpha 1 activity is similar to the modulation induced by beta 1, beta 2, or beta 3. The most striking effect of beta subunits is their ability to increase functional alpha 1 activity, which can be measured as either increased dihydropyridine binding to membranes from transfected COS cells or increased calcium channel activity in Xenopus oocytes.