Cholesterol (Chol) localizes predominantly in the lipid rafts and caveolae in the membrane and regulates functions of ion channels. In the aortic smooth muscle cells, Ca2+ influx to initiate contraction is supplied mainly by window ICa,L that is determined by the steady-state activation and inactivation of the channel. We clarified the effect of overload and depletion of Chol on both the activation and the inactivation of ICa,L. Previous studies have reported that the overload of Chol utilizing methyl-β-cyclodextrin (CD): cholesterol complex or Chol dissolved by ethanol induces strong inhibition of ICa,L. We applied water-soluble PEG600: cholesterol (PC) to overload Chol and used CD to deplete Chol. Cultured A7r5, a cell line from fetal rat aorta, cells were dissociated and incubated with normal Tyrode solution containing PC or CD in micro-tubes for longer than 90 min at room temperature. In some experiments CD was added after the pretreatment by PC to examine its reversibility. ICa,L was recorded as whole-cell current with 10 mM Ba2+ as the charge carriers and small T-type Ca2+ channel currents were inactivated by 50 ms pre-pulse to -40 mV. The maximal ICa,L density (ICa,L,max) was obtained at 0 mV and was 6.8±0.8 pA/pF (mean±s.e.m., n=18) in the time-matched control. The pretreatment with PC inhibited ICa,L in a dose-dependent manner without any voltage-shift of the I-V relationship. The ICa,L,max after the incubation with PC was: 0.1 mM, 4.9±0.7 (n=10, p<0.05 compared with control), 1 mM, 3.1±0.6 (11, p<0.001) and 10 mM, 2.8±0.2 pA/pF (14, p<0.001). It was significantly increased by the pretreatment with CD: 10 mM, 8.7±1.2 (n=14, p<0.05) and 30 mM, 9.5±1.2 pA/pF (n=13, p<0.001). We found that loading of Chol by PC augments the voltage-dependent inactivation of ICa,L. V0.5 for the steady-state inactivation (f-V) curve was: control, -30.1±0.5 mV (n=24); 0.1 mM-PC, -32.6±0.5 mV (n=10); 1 mM, -38.0±0.3 mV (n=15); 10 mM, -42.1±0.3 mV (n=14). On the other hand, the depletion of Chol by CD shifted V0.5 to a depolarizing direction: 10 mM, -26.0±0.6 mV (n=19) and 30 mM, -21.7±0.8 mV (n=20). The addition of 30 mM CD to 1 mM PC-pretreated cells in the tube hardly reversed the PC-induced leftward shift of f-V relationship: V0.5, -35.6±0.4 mV (n=11). It implicates that PC could not be removed by CD from the membrane. However, the addition of 30 mM CD after the overload of Chol by 1 mM-PC augmented ICa,L,max to -12.7±1.6 pA/pF (n=11, p<0.001 compared with 30 mM CD alone at -10 mV) and shifted the I-V curve to the left. We conclude that Chol is involved in the regulation of basal window ICa,L by decreasing the current density and by augmenting the voltage-dependent inactivation. Chol may be also involved in the regulation of cellular signaling to modulate ICa,L.