Synaptic transmission is a well-orchestrated sequence of processes, which take place both at presynaptic and postsynaptic membranes. Therefore, in addition to the many proteins involved in synaptic transmission, plasma membrane lipids have to be studied as potential modulators of synaptic transmission. In this study, we performed a comprehensive analysis of the role of cholesterol in glutamatergic synaptic transmission. Using cultured hippocampal and cerebellar neurons and their acute cholesterol depletion by cyclodextrine, we found that cholesterol is important for both presynaptic and postsynaptic steps of synaptic transmission. Our data show that cholesterol is essential for the function of NMDA receptors and for keeping NMDA receptors localized in postsynaptic membrane. Regarding the presynaptic effects of cholesterol, our experiments indicate an essential role of cholesterol for action potential propagation along the axon and an inhibitory effect of cholesterol on glutamate release from presynaptic terminals.