In this study we tested the hypothesis that ventricular homeostasis of L-type Ca²⁺ current (I_Ca,L) minimally involves regulation of the main pore forming -subunit (Ca_V1.2) and auxiliary proteins that serve as positive or negative regulators of I_Ca,L. We treated animals for 24 hours with verapamil (VER, 3.6mg/kg/day) isoproterenol (ISO, 30mg/kg/day), or VER +ISO via osmotic minipumps. To test for alterations of Ca²⁺ channel complex components we performed real time-PCR and western blot analysis on ventricle. In addition, CMs were dispersed and current was recorded in the whole-cell configuration to evaluate I_Ca,L. Surprisingly, 24-48 hour VER increased Ca_V1.2 mRNA and protein, and I_Ca,L current (VER: 11+/-1pA/pF vs control:7+/-0.5pA/pF, p<0.01). I_Ca,L from CMs in VER mice showed no change in whole-cell capacitance. To examine the in vivo effects of a physiologically-relevant Ca channel agonist we treated mice with ISO. 24 h ISO infusion increased heart rate; Ca_V1.2- and CaV2 mRNA levels were constant, but the Ca²⁺ channel subunit mRNA Rem was increased 2-fold. Cells isolated from 24 hour ISO hearts showed no change in basal I_Ca,L density, and diminished responsiveness to acute 1 µM ISO. To further examine the homeostatic regulation of the Ca channel, we treated animals for 24 hours with a combination of VER and ISO. The influence of VER+ISO was similar to ISO alone on Ca²⁺ channel mRNAs and I_Ca,L with the exception that it prevented the increase in Rem seen with ISO treatment. Long-term Ca²⁺ channel blockade induces an increase of Ca_V1.2 mRNA and protein and significantly increased I_Ca,L.