<p>With a multitude of substrates, &gamma;-secretase is poised to control neuronal function through a variety of signaling pathways. Presenilin 1 (PS1) is an integral component of &gamma;-secretase and is also a protein closely linked to the etiology of Alzheimer&#39;s disease (AD). To better understand the roles of &gamma;-secretase and PS1 in normal and pathological synaptic transmission, we examined evoked and spontaneous neurotransmitter release in cultured hippocampal neurons derived from PS1 knock-out (KO) mice. We found no changes in the size of evoked synaptic currents, short-term plasticity, or apparent calcium dependence of evoked release. The rate of spontaneous release from PS1 KO neurons was, however, approximately double that observed in wild-type (WT) neurons. This increase in spontaneous neurotransmission depended on calcium influx but did not require activation of voltage-gated calcium channels or presynaptic NMDA receptors or release of calcium from internal stores. The rate of spontaneous release from PS1 KO neurons was significantly reduced by lentivirus-mediated expression of WT PS1 or familial AD-linked M146V PS1, but not the D257A PS1 mutant that does not support &gamma;-secretase activity. Treatment of WT neuronal cultures with &gamma;-secretase inhibitor mimicked the loss of PS1, leading to a selective increase in spontaneous release without any change in the size of evoked synaptic currents. Together, these results identify a novel role for &gamma;-secretase in the control of spontaneous neurotransmission through modulation of low-level tonic calcium influx into presynaptic axon terminals.</p>