To maximize the speed of information processing, some synapses can sustain high-frequency transmission. However, the limited number of synapses allowing direct recordings has hampered our understanding of the mechanisms of high-frequency synaptic transmission. Here, we establish two-photon microscopy guided recordings from fluorescence labeled presynaptic cerebellar mossy fiber boutons (cMFBs) paired with recordings from postsynaptic granule cells to analyze high-frequency signaling. Remote stimulation of the mossy fiber axon can elicit action potentials at >1 kHz frequency in cMFBs with mean half-width of 123 ± 9 µs (n = 11) and minimal action potential broadening during high-frequency activation. Paired recordings show that frequencies as high as 1 kHz can indeed be transmitted at these synapses. Furthermore, deconvolution of postsynaptic currents in combination with presynaptic capacitance measurements indicates heterogeneous release probabilities and rapid vesicle reloading kinetics. Finally, elevation of the calcium buffer EGTA to 5 mM in the presynaptic terminal had little effect on the initial fast release component, indicating tight coupling between calcium channels and sensors of exocytosis in a subset of vesicles. These data suggest that a variety of functional specializations permits kHz-transmission at a central synapse.