Synaptic strength is determined by release probability and the size of the readily releasable pool of docked vesicles. Here we describe the effects of blocking myosin light chain kinase (MLCK), a cytoskeletal regulatory protein thought to be involved in myosin-mediated vesicle transport, on synaptic transmission at the mouse calyx of Held synapse. Application of three different MLCK-inhibitors increased the amplitude of the early EPSCs in a stimulus train, without affecting the late steady-state EPSCs. A presynaptic locus of action for MLCK-inhibitors was confirmed by an increase in the frequency of miniature EPSCs that left their average amplitude unchanged. MLCK-inhibition did not affect presynaptic Ca²⁺ currents, or action potential waveform. Moreover, Ca²⁺ imaging experiments showed that [Ca²⁺]_i transients elicited by 100 Hz stimulus trains were not altered by MLCK-inhibition. Studies using high frequency stimulus trains indicated that MLCK-inhibitors increase vesicle pool size, but do not alter significantly release probability. Accordingly, when AMPA receptor desensitization was minimized, EPSC paired-pulse ratios were unaltered by MLCK-inhibition, suggesting that release probability remains unaltered. MLCK-inhibition potentiated EPSCs even when presynaptic Ca²⁺ buffering was greatly enhanced by treating slices with EGTA-AM. In addition, MLCK-inhibition did not affect the rate of recovery from short-term depression. Finally, developmental studies revealed that EPSC potentiation by MLCK-inhibition starts at postnatal day 5 (P5), and remains strong during synaptic maturation up to P18. Overall, our data suggest that MLCK plays a crucial role in determining the size of the pool of synaptic vesicles that undergo fast release at a CNS synapse.