Neurotransmitter release displays at least two kinetically distinct components in response to a single action potential. The majority of release occurs synchronously with action-potential-triggered Ca(2+) influx; however, delayed release--also called asynchronous release--persists for tens of milliseconds following the peak Ca(2+) transient. In response to trains of action potentials, synchronous release eventually declines, whereas asynchronous release often progressively increases, an effect that is primarily attributed to the buildup of intracellular Ca(2+) during repetitive stimulation. The precise relationship between synchronous and asynchronous release remains unclear at central synapses. To gain better insight into the mechanisms that regulate neurotransmitter release, we systematically characterized the two components of release during repetitive stimulation at excitatory autaptic hippocampal synapses formed in culture. Manipulations that increase the Ca(2+) influx triggered by an action potential--elevation of extracellular Ca(2+) or bath application of tetraethylammonium (TEA)--accelerated the progressive decrease in synchronous release (peak excitatory postsynaptic current amplitude) and concomitantly increased asynchronous release. When intracellular Ca(2+) was buffered by extracellular application of EGTA-AM, initial depression of synchronous release was equal to or greater than control; however, it quickly reached a plateau without further depression. In contrast, asynchronous release was largely abolished in EGTA-AM. The total charge transfer following each pulse--accounting for both synchronous and asynchronous release--reached a steady-state level that was similar between control and EGTA-AM. A portion of the decreased synchronous release in control conditions therefore was matched by a higher level of asynchronous release. We also examined the relative changes in synchronous and asynchronous release during repetitive stimulation under conditions that highly favor asynchronous release by substituting extracellular Ca(2+) with Sr(2+). Initially, asynchronous release was twofold greater in Sr(2+). By the end of the train, the difference was approximately 50%; consequently, the total release per pulse during the plateau phase was slightly larger in Sr(2+) compared with Ca(2+). We thus conclude that while asynchronous release--like synchronous release--is limited by vesicle availability, it may be able to access a slightly larger subset of the readily releasable pool. Our results are consistent with the view that during repetitive stimulation, the elevation of asynchronous release depletes the vesicles immediately available for release, resulting in depression of synchronous release. This implies that both forms of release share a small pool of immediately releasable vesicles, which is being constantly depleted and refilled during repetitive stimulation.