Modulation of the size of the readily releasable vesicle pool has recently come under scrutiny as a candidate for the regulation of synaptic strength. Using electrophysiological and optical measurement techniques, we show that phorbol esters increase the size of the readily releasable pool at glutamatergic hippocampal synapses in culture through a protein kinase C (PKC)-dependent mechanism. Phorbol ester activation of PKC also increases the rate at which the pool refills. These results identify two powerful ways that activation of the PKC pathway may regulate synaptic strength by modulating the readily releasable pool of vesicles.