Caveolae and transverse (T-) tubules are membrane structures enriched in cholesterol and glycosphingolipids. They play an important role in receptor signalling and myogenesis. The T-system is also highly enriched in dihydropyridine receptors (DHPRs), which control excitation-contraction (E-C) coupling. Recent results have shown that a depletion of membrane cholesterol alters caveolae and T-tubules, yet detailed functional studies of DHPR expression are lacking. Here we studied electrophysiological and morphological effects of methyl--cyclodextrin (MCD), a cholesterol-sequestering drug, on freshly isolated foetal skeletal muscle cells. Exposure of foetal myofibres to 1-3 mM MCD for 1 hour at 37°C led to a significant reduction in caveolae and T-tubule areas and to a decrease in cell membrane electrical capacitance. In whole-cell voltage-clamp experiments, the L-type Ca2+ current amplitude was significantly reduced, and its voltage-dependence was shifted ~15 mV toward more positive potentials. Activation and inactivation kinetics were slower in treated cells than in control cells and stimulation by a saturating concentration of Bay K 8644 was enhanced. In addition, intramembrane charge movement and Ca2+ transients evoked by a depolarisation were reduced without shift of the midpoint, indicating a weakening of E-C coupling. In contrast, T-type Ca2+ current was not affected by MCD treatment. Most of the L-type Ca2+ conductance reduction and E-C coupling weakening could be explained by a decrease of the number of DHPRs due to the disruption of caveolae and T-tubules. However, the effects on L-type channel gating kinetics suggest that membrane cholesterol content modulates DHPR function. Moreover, the significant shift of the voltage-dependence of L-type current without change in the voltage-dependence of charge movement and Ca2+ transients suggests that cholesterol differentially regulates the two functions of the DHPR.