Endocytosis is an important step in the synaptic vesicle recycling; it helps to maintain reliable neurotransmitter release. Two major pathways of endocytosis co-exist in the same presynaptic terminal: clathrin-dependent endocytosis of single vesicles utilizing adaptor protein AP-2 and bulk endocytosis leading to the formation of endosomes and dependent on adaptor protein AP-3. Vesicles derived in these two pathways differ in their molecular content and represent distinct vesicular pools. We used AP-3 knockout mice (Ap3b2-/-) lacking endosomal vesicle formation to explore physiological role of vesicular pool formed via this pathway in synapses between mossy fiber boutons and hippocampal CA3 pyramidal cell. Spontaneous and evoked EPSCs had similar amplitude and kinetic properties, moreover quantal parameters (p, Q and N), ready releasable pool size and its recovery speed were comparable between WT and KO mice. However, in KOs decay time constant of release was significantly faster (3.7 ± 0.4 ms vs 5 ±0.4 ms) and was accompanied with reduced asynchronous release (3.8 vs 7.4 Hz). In addition, stimulation with natural spike trains revealed significantly reduced probability of postsynaptic APs and increased variability of responses, especially prominent at ~100 Hz increase of stimulation frequency. Our data suggest that vesicles generated via bulk endocytosis contribute primarily to asynchronous release. The lack of asynchronous release renders information transfer from granule cells to hippocampal pyramidal cells less reliable, and therefore it could influence spatial memory.