Recombinant adenoviruses were used to overexpress green fluorescent protein (GFP)‐fused auxiliary Ca²⁺ channel β subunits (β₁‐β₄) in cultured adult rat heart cells, to explore new dimensions of β subunit functions in vivo. Distinct β‐GFP subunits distributed differentially between the surface sarcolemma, transverse elements, and nucleus in single heart cells. All β‐GFP subunits increased the native cardiac whole‐cell L‐type Ca²⁺ channel current density, but produced distinctive effects on channel inactivation kinetics. The degree of enhancement of whole‐cell current density was non‐uniform between β subunits, with a rank order of potency β_2aαβ₄ > β_1b > β₃. For each β subunit, the increase in L‐type current density was accompanied by a correlative increase in the maximal gating charge (Q_max) moved with depolarization. However, β subunits produced characteristic effects on single L‐type channel gating, resulting in divergent effects on channel open probability (P_o). Quantitative analysis and modelling of single‐channel data provided a kinetic signature for each channel type. Spurred on by ambiguities regarding the molecular identity of the actual endogenous cardiac L‐type channel β subunit, we cloned a new rat β₂ splice variant, β_2b, from heart using 5′ rapid amplification of cDNA ends (RACE) PCR. By contrast with β_2a, expression of β_2b in heart cells yielded channels with a microscopic gating signature virtually identical to that of native unmodified channels. Our results provide novel insights into β subunit functions that are unattainable in traditional heterologous expression studies, and also provide new perspectives on the molecular identity of the β subunit component of cardiac L‐type Ca²⁺ channels. Overall, the work establishes a powerful experimental paradigm to explore novel functions of ion channel subunits in their native environments.