The synaptic transmission at hippocampal mossy fiber (MF) synapses is modulated by protein kinase C (PKC) at the presynaptic terminal. The mechanisms of this modulation were investigated at individual MF terminals. We used a TV-42 transgenic mouse line that expresses synaptopHluorin (SpH), one of optical probes measuring exo-endocytosis, specifically in the hippocampal mossy fiber terminals. The mouse was deeply ether-anesthetized and the brain was removed. The hippocampal slices were made at 400 μm. Individual large mossy fiber terminals were identified under confocal microscopy and the activity-dependent changes of SpH fluorescence were measured. The activity-dependent change of SpH fluorescence was on average increase by phorbol esters, synthetic analogues of diacylglycerol, whereas not by 4α-phorbol, one of inactive analogues. Phorbol esters increased the fusion probability of a vesicle in some boutons (type 1), whereas they preferentially increase the readily releasable pool in others (type 2). Some other synapses are presynaptically silent but become releasable upon exposure to phorbol esters (type 3). These effects were almost completely inhibited by staurosporine, one of PKC antagonists. Thus the PKC-dependent exocytosis modulation is variable from MF synapse to synapse. All animal procedures were conducted in accordance with the guiding principles of Physiological Society of Japan. [J Physiol Sci. 2007;57 Suppl:S154]