In cardiomyocytes, the L-type calcium channel inactivation processes (voltage- and calcium-dependent; VDI and CDI respectively) has been associated with the determination of the action potential duration. Interestingly, in rat cardiomyocytes although the β-adrenergic stimulation has been shown to increase L-type calcium current when studied under square pulses, the action potential duration is reduced. In this work, we explored this apparent contradiction combining self-action potential clamp (sAP-Clamp) with the molecular inhibition of VDI or CDI (by overexpressing CaVβ2a or calmodulin mutants respectively) to directly record the L-type calcium current during the action potential in newborn rat cardiomyocytes. Our results show that, in control cardiomyocytes, L-type current coincides with the action potential kinetic, and while the inhibition of either inactivation processes prolongs the action potential duration, the L-type current extends its duration accordingly, suggesting that both processes are important in controlling L-type current during an action potential. In contrast, after β-adrenergic stimulation, L-type calcium current finishes before than the action potential, same effect observed in cells where the L-type channel has a reduced VDI, but not in cells without CDI, where the L-type current maintains the same kinetic than the action potential, demonstrating experimentally that the CDI is the inactivation process that governs the L-type current kinetic during β-adrenergic stimulation.