Ca2+ buffer saturation was proposed as a mechanism of paired pulse facilitation (PPF). However, whether it operates under native conditions remained unclear. Here we show that saturation of the endogenous fast Ca2+ buffer calbindin-D28k (CB) plays a major role in PPF at CB-containing synapses. Paired recordings from synaptically connected interneurons and pyramidal neurons in the mouse neocortex revealed that dialysis increased the amplitude of the first response and decreased PPF. Loading the presynaptic terminals with BAPTA or CB rescued the effect of the CB washout. We extended the study to the CB-positive facilitating excitatory mossy fiber-CA3 pyramidal cell synapse. The effects of different extracellular Ca2+ concentrations and of EGTA indicated that PPF in CB-containing terminals depended on Ca2+ influx rather than on the initial release probability. Experiments in CB knockout mice confirmed that buffer saturation is a novel basic presynaptic mechanism for activity-dependent control of synaptic gain.