The beta subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming alpha1 subunits. The beta3 subunit is expressed in the adrenal gland and participates in forming various calcium channel types. We performed a series of experiments in beta3-null mice to determine the role of the beta3 subunit in catecholamine release from the adrenal chromaffin system. Protein levels of N-type channel forming CaV2.2 and L-type forming CaV1.2 decreased. The beta3-null mice showed a decreased baroreflex, suggesting decreased sympathetic tonus, whereas plasma catecholamine levels did not change. Pulse-voltage stimulation revealed significantly increased amperometrical currents in beta3-null mice, while patch-clamp recordings showed a significant reduction in Ca(2+)-currents due to reduced L- and N-type currents, indicating facilitated exocytosis. A biochemical analysis revealed increased InsP3 production. In conclusion, our results indicate the importance of the beta3 subunit in determining calcium channel characteristics and catecholamine release in adrenal chromaffin cells.