This study examines a form of short-term depression (STD) at excitatory central synapses that persists for several minutes after induction, and corresponds to the slowest rate-limiting step in the synaptic vesicle cycle. After initial depletion of the readily releasable pool (RRP) of synaptic vesicles, extended trains of 20 Hz stimulation drove exocytosis to a maximal steady state rate of less than 1 quantum per synapse per second - although substantially higher release rates could be elicited at rested synapses. Induction of STD under these conditions proceeded in two phases. The first has been attributed to RRP depletion in previous studies; the second is shown here to reflect a progressive reduction in the overall rate at which vesicles are readied for release, and is termed "supply-rate depression." Supply-rate depression is identified further with a rate-limiting step in the synaptic vesicle cycle that emerges during extended episodes of heavy use to slow the time course of RRP replenishment via conversion from a fast to a slow vesicle re-supply mechanism, with exponential time constants of ~7 s and ~1 min, respectively. The onset of supply-rate depression occurred with a marked delay of several seconds at Schaffer collateral synapses, but substantially more quickly at excitatory synapses of the visual cortex. The delay is not compatible with previous models of STD, but is well fitted by a novel model presented here. Supply-rate depression may be important for limiting synaptic throughput under conditions of heavy use, and could thus function as a native anti-epilepsy device.