Background and purpose Exosomes, nanosized extracellular vesicles, emerged as players in the cell-to-cell communication in the central nervous system (CNS), having a role in the modulation of the synaptic activity. This study aimed at evaluating whether exosomes can be actively released from presynaptic nerve terminals. Experimental Approach Mouse cortical synaptosomes were exposed to a depolarizing stimulus (25 mM KCl medium) and exosomes were isolated from the synaptosomal supernatants. Exosomes were characterized by dynamic light scattering, transmission electron microscopy, western blot and flow cytometry analyses. We also evaluated whether and how removing external Ca2+ ions or activating presynaptic GABAB receptors by exposing synaptosomes to (±)-baclofen (10 µM) affects the 25 mM KCl-evoked release of exosomes. Key Results The structural and biochemical analysis unveiled that synaptosomal supernatants contained vesicles having the size and the shape of exosomes, immunopositive for the exosomal markers TSG101, flotillin-1, CD63 and CD9. The content of these proteins in the exosomal fraction isolated from synaptosomal supernatants increased upon the exposure of nerve terminals to a depolarizing stimulus and occurred in a Ca2+-dependent fashion, mimicking the release of glutamate. (±)-Baclofen significantly reduced glutamate exocytosis but failed to affect the release of exosomes from cortical synaptosomes. Finally, presynaptic exosomes were shown to carry selected NMDA and AMPA receptors subunits. Conclusion and implications Our study unveils the Ca2+-dependent, depolarization-evoked release of exosomes from mouse cortical nerve terminals, which is insensitive to presynaptic release-regulating GABAB receptors. These findings add new insights into the mechanisms of exosomes-mediated communication in CNS.