The readily releasable pool (RRP) of vesicles is a core concept in studies of presynaptic function. However, operating principles lack consensus definition and the utility for quantitative analysis has been questioned. Here we confirm that RRPs at calyces of Held from 14 to 21 day old mice have a fixed capacity for storing vesicles that is not modulated by Ca2+. Discrepancies with previous studies are explained by a dynamic flow-through pool, established during heavy use, containing vesicles that are released with low probability despite being immediately releasable. Quantitative analysis ruled out a posteriori explanations for the vesicles with low release probability, such as Ca2+-channel inactivation, and established unexpected boundary conditions for remaining alternatives. Vesicles in the flow-through pool could be incompletely primed, in which case the full sequence of priming steps downstream of recruitment to the RRP would have an average unitary rate of at least 9/s during heavy use. Alternatively, vesicles with low and high release probability could be recruited to distinct types of release sites, in this case the timing of recruitment would be similar at the two types, and the downstream transition from recruited to fully primed would be much faster. In either case, further analysis showed that activity accelerates the upstream step where vesicles are initially recruited to the RRP. Overall, our results show that the RRP can be well defined in the mathematical sense, and support the concept that the defining mechanism is a stable group of autonomous release sites. 