The amount of neurotransmitter stored in synaptic vesicles determines postsynaptic quantal size and thus the strength of synaptic transmission. However, little is known about regulation of vesicular neurotransmitter uptake. In recordings from the calyx of Held, a giant mammalian glutamatergic synapse, we found that changes in presynaptic Na(+) concentration above and below a resting value of 13 mM regulated vesicular glutamate uptake, consistent with activation of a vesicular monovalent cation Na(+)(K(+))/H(+) exchanger. Na(+) flux through presynaptic plasma membrane hyperpolarization-activated cyclic nucleotide-gated (HCN) channels enhanced presynaptic Na(+) concentration and thus controlled postsynaptic quantal size. Our results indicate that a plasma membrane ion channel controls synaptic strength by modulating vesicular neurotransmitter uptake through a Na(+)-dependent process.