The β subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming α1 subunits. Of the four β subunits reported to date, the β3 subunit is highly expressed in smooth muscle cells and is thought to consist of L-type calcium channels. To determine the role of the β3 subunit in the voltage-dependent calcium channels of the cardiovascular system in situ, we performed a series of experiments in β3-null mice. Western blot analysis indicated a significant reduction in expression of the α1 subunit in the plasma membrane of β3-null mice. Dihydropyridine binding experiments also revealed a significant decrease in the calcium channel population in the aorta. Electrophysiological analyses indicated a 30% reduction in Ca2+ channel current density, a slower inactivation rate, and a decreased dihydropyridine-sensitive current in β3-null mice. The reductions in the peak current density and inactivation rate were reproduced in vitro by co-expression of the calcium channel subunits in Chinese hamster ovary cells. Despite the reduced channel population, β3-null mice showed normal blood pressure, whereas a significant reduction in dihydropyridine responsiveness was observed. A high salt diet significantly elevated blood pressure only in the β3-null mice and resulted in hypertrophic changes in the aortic smooth muscle layer and cardiac enlargement. In conclusion, this study demonstrates the involvement and importance of the β3 subunit of voltage-dependent calcium channels in the cardiovascular system and in regulating channel populations and channel properties in vascular smooth muscle cells.