Glycolytic ATP synthesis by synaptic vesicles provides an efficient mechanism for fueling vesicular loading of the neurotransmitter glutamate. This is achieved in part by vesicle-bound pyruvate kinase. However, we have found that vesicular glutamate uptake, in the presence of the pyruvate kinase substrates ADP and phosphoenolpyruvate (PEP), substantially exceeds that caused by exogenous ATP. We propose that this much enhanced uptake is in part due to extra ATP produced via a mechanism involving a novel enzyme, PEP-dependent ADP synthase. We discuss implications for this enzyme in energy homeostasis and pathophysiology, as well as in efficient synaptic glutamate transmission.