Cholesterol is a primary constituent of the plasmalemma, including the lipid rafts/caveolae where various G protein-coupled receptors colocalize with signaling proteins and channels. By manipulating cholesterol in rabbit and rat ventricular myocytes using methyl--cyclodextrin (MCD), we studied the role of cholesterol in the modulation of L-type Ca²⁺ currents (I_Ca,L). MCD was mainly dialyzed from BAPTA-containing pipette solution during whole-cell clamp. In rabbit myocytes dialyzed with 30 mM MCD for 10 min, a positive shift in V_0.5 (membrane potential at half-maximal activation) from -8 to -2 mV developed and was associated with an increase in current density at positive potentials (42% at +20 mV vs. time-matched controls). Isoproterenol (ISO) increased I_Ca,L ~3-fold and caused a negative shift in V0.5 in control cells, but it did not increase I_Ca,L in MCD-treated myocytes, nor did it shift the V_0.5. The effect of MCD (10 or 30 mM) was concentration dependent, so that 30 mM MCD suppressed the ISO-induced increase in I_Ca,L more effectively than 10 mM MCD. MCD dialysis also abolished the increase in I_Ca,L elicited by forskolin or dibutyryl-cAMP, but not that elicited by (-)BAY K8644. External application of MCD:cholesterol complex to rat myocytes attenuated the MCD-mediated inhibition of the ISO-induced increase of I_Ca,L. Biochemical analysis confirmed that the myocytes’cholesterol content was diminished by MCD and increased by its cholesterol-loaded complex. Cholesterol thus appears to contribute to the regulation of both basal ICa,L and -adrenergic cAMP/PKA-mediated increases in I_Ca,L. We suggest that cholesterol affects the structural coupling between L-type Ca²⁺ channels and adjacent regulatory proteins.