The L-type Ca2+ channel (LTCC) appears to be localised predominantly to the t-tubule membrane in mammalian cardiac ventricular myocytes. The mechanism of this localisation is unknown, but in other cell types it has been shown that t-tubule membrane is lipid-rich [1], and that the α2δ subunit of the LTCC partitions into lipid-rich membrane fractions [2]. In the present study, we investigated the effect of methyl-β-cyclodextrin (MβCD), which decreases membrane cholesterol content [3, 4], on the distribution and function of the LTCC. Adult rat ventricular myocytes were isolated and either treated with MβCD (1 mM, 60 min, 37°C or 5 mM, 120 min, room temperature) or kept as time- and temperature-matched controls. In cells stained with di-8-ANEPPS and imaged using confocal microscopy, treatment with MβCD appeared to have no significant effect on t-tubule structure, assessed using fast Fourier transform (FFT) of the transverse staining pattern. Immunocytochemical studies showed no significant change in the intensity or total area of staining of ryanodine receptors (RyR) or of the α1C or α2δ-1 subunits of the LTCC. However, FFT analysis of the transverse staining pattern showed that the distribution of the α1C subunit and RyR was significantly disrupted (P = 0.039 and P = 0.019 respectively, paired t-test), whereas the staining pattern of the α2δ-1 subunit appeared unaffected (P > 0.05). The whole-cell patch clamp technique was used to monitor L-type Ca2+ current (ICa). From a holding potential of -80 mV, a depolarising pre-pulse to -40 mV (to inactivate fast Na+ current) was followed by test pulses to a range of voltages between -30 and +50 mV. Treatment with MβCD significantly decreased peak ICa density (from -9.55 ± 0.43 to -7.14 ± 0.42 pA/pF at 0 mV, mean ± S.E.M.; n = 12/9; P < 0.001, unpaired t-test), with no change to half-maximal activation voltage. This resulted from a significant decrease of ICa (from -1.51 ± 0.16 to -0.86 ± 0.14 nA; P = 0.008) and a non-significant decrease in membrane capacitance (from 157 ± 12.3 to 121 ± 21.5 pF). These data suggest that depletion of membrane cholesterol disrupts the distribution of LTCC and RyR and decreases the number of functional Ca2+ channels in the cell membrane, and thus that membrane cholesterol plays a role in the localisation of LTCC in the cell membrane.