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Cholesterol is a key component of the cell plasma membrane. It has been suggested that the t-tubule membrane of cardiac ventricular myocytes is enriched in cholesterol, and that this plays a role in determining t-tubule structure and function. We have used methyl-β-cyclodextrin (MβCD) to deplete cholesterol in intact and detubulated mouse ventricular myocytes to investigate the contribution of cholesterol to t-tubule structure, membrane capacitance, and the distribution of Ca flux pathways. Depletion of membrane cholesterol was confirmed using filipin, however di-8-ANEPPS staining showed no differences in t-tubule structure following MβCD-treatment. MβCD-treatment had no significant effect on the capacitance:volume relationship of intact myocytes, or on the decrease in capacitance:volume caused by detubulation. Similarly, Ca influx and efflux were not altered by MβCD-treatment, and were reduced by a similar amount following detubulation in untreated and MβCD-treated cells. These data show that cholesterol depletion has similar effects on the surface and t-tubule membranes, and suggest that cholesterol plays no acute role in determining t-tubule structure and function.