Mammalian motor nerve terminals contain hundreds of thousands of synaptic vesicles, but only a fraction of these vesicles is immediately available for release, the remainder forming a reserve pool. The supply of vesicles is replenished through endocytosis, and newly formed vesicles are refilled with acetylcholine through a process that depends on the vesicular acetylcholine transporter (VAChT). During expression of short-term plasticity, quantal release can be increased, but it is unknown whether this reflects enhanced recruitment of vesicles from the reserve pool or rapid recycling. We examined spontaneous and evoked release of acetylcholine at endplates from genetically modified VAChT KD(HOM) mice that express approximately 30% of the normal level of VAChT to determine steps rate-limited by synaptic vesicle filling. Quantal content and quantal size were reduced in VAChT KD(HOM) mice compared with wild-type controls. Although high-frequency stimulation did not reduce quantal size further, the post-tetanic increase in end-plate potential amplitude or MEPP frequency was significantly smaller in VAChT KD(HOM) mice. This was the case even when tetanic depression was eliminated using an extracellular solution containing reduced Ca(2+) and raised Mg(2+). These results reveal the dependence of short-term plasticity on the level of VAChT expression and efficient synaptic vesicle filling.