Regulation of ionic channels plays a pivotal role in controlling cardiac function. Here we show that Rho family of small G-proteins regulate L-type Ca²⁺ currents in ventricular cardiomyocytes. Ventricular myocytes isolated from transgenic (TG) mice overexpressing Rho GDI, a specific GDP dissociation inhibitor, exhibited a significantly decreased basal L-type Ca²⁺ current density (about 40%) compared with myocytes from nontransgenic (NTG) mice. A Ca²⁺ channel agonist, Bay K, and a -adrenergic agonist, isoproterenol, increased Ca²⁺ currents in both NTG and TG myocytes to a similar maximal level, and no change in mRNA and protein level of the 1 subunit of the Ca²⁺ channel was observed. These results suggest that the channel activity, but not the expression levels, was altered in TG myocytes. In addition, the densities of inward rectifier K⁺ currents and the transient outward K⁺ currents were unchanged in TG myocytes. The amplitudes and rates of basal twitch and Ca²⁺ transients were also similar between the two groups. When the protein was directly delivered into adult ventricular myocytes via TAT-mediated protein transduction, Rho GDI significantly decreased Ca²⁺ current, supporting that the defect of Ca²⁺ channel activity in TG myocytes was a primary effect of the transgene. In addition, expression of a dominant negative RhoA, but not a dominant negative Rac1 or Cdc42, also significantly decreased Ca²⁺ current, indicating that inhibition of Ca²⁺ channel activity by overexpression of Rho GDI is mediated by inhibition of RhoA. This study points to the L-type Ca²⁺ channel activity as a novel downstream target of the RhoA signaling pathway.