Increases in Ca²⁺ influx through the L-type Ca²⁺ channel (LTCC, Cav1.2) augment sarcoplasmic reticulum (SR) Ca²⁺ loading and the amplitude of the cytosolic Ca²⁺ transient to enhance cardiac myocyte contractility. Our hypothesis is that persistent increases in Ca²⁺ influx through the LTCC cause apoptosis if the excessive influx results in SR Ca²⁺ overload. Feline ventricular myocytes (VMs) in primary culture were infected with either an adenovirus (Ad) containing a rat Cav1.2 β_2a subunit-green fluorescent protein (GFP) fusion gene (Adβ_2a) to increase Ca²⁺ influx or with AdGFP as a control. Significantly fewer β_2a-VMs (21.4±5.6%) than GFP-VMs (99.6±1.7%) were viable at 96 hours. A fraction of β_2a-VMs (20.8±1.8%) contracted spontaneously (SC-β_2a-VMs), and viability was significantly correlated with the percentage of SC-β_2a-VMs. Higher percentages of apoptotic nuclei, DNA laddering, and cytochrome C release were detected in β_2a-VMs. This apoptosis was prevented with pancaspase or caspase-3 or caspase-9 inhibitors. L-type calcium current (I_Ca-L) density was greater in β_2a-VMs (23.4±2.8 pA/pF) than in GFP-VMs (7.6±1.6 pA/pF). SC-β_2a-VMs had higher diastolic intracellular Ca²⁺ (Indo-1 ratio: 1.1±0.1 versus 0.7±0.03, P<0.05) and systolic Ca²⁺ transients (1.89±0.27 versus 0.80±0.08) than GFP-VMs. Inhibitors of Ca²⁺ influx, SR Ca²⁺ uptake and release, mitochondrial Ca²⁺ uptake, mitochondrial permeation transition pore, calpain, and Bcl-2-associated X protein protected β_2a-VMs from apoptosis. These results show that persistent increases in Ca²⁺ influx through the I_Ca-L enhance contractility but lead to apoptosis through a mitochondrial death pathway if SR Ca²⁺ overload is induced.