Sequence analysis of the human genome permitted cloning of five Ca2+-channel β2 splice variants (β2a–β2e) that differed only in their proximal amino-termini. The functional consequences of such β2-subunit diversity were explored in recombinant L-type channels reconstituted in HEK 293 cells. β2a and β2e targeted autonomously to the plasma membrane, whereas β2b–β2d localized to the cytosol when expressed in HEK 293 cells. The pattern of modulation of L-type channel voltage-dependent inactivation gating correlated with the subcellular localization of the component β2 variant—membrane-bound β2a and β2e subunits conferred slow(er) channel inactivation kinetics and displayed a smaller fraction of channels recovering from inactivation with fast kinetics, compared to β2b–β2d channels. The varying effects of β2 subunits on inactivation gating were accounted for by a quantitative model in which L-type channels reversibly distributed between fast and slow forms of voltage-dependent inactivation—membrane-bound β2 subunits substantially decreased the steady-state fraction of fast inactivating channels. Finally, the β2 variants also had distinctive effects on L-type channel steady-state activation gating, as revealed by differences in the waveforms of tail-activation (G-V) curves, and conferred differing degrees of prepulse facilitation to the channel. Our results predict important physiological consequences arising from subtle changes in Ca2+-channel β2-subunit structure due to alternative splicing and emphasize the utility of splice variants in probing structure-function mechanisms.