Mutations in PRoline-Rich Transmembrane protein 2 (PRRT2) underlie a group of paroxysmal disorders including epilepsy, kinesigenic dyskinesia and migraine. Most of the mutations lead to impaired PRRT2 expression and/or function, emphasizing the pathogenic role of the PRRT2 deficiency. In this work, we investigated the phenotype of primary hippocampal neurons obtained from mouse embryos in which the PRRT2 gene was constitutively inactivated. Although PRRT2 is expressed by both excitatory and inhibitory neurons, its deletion decreases the number of excitatory synapses without significantly affecting the number of inhibitory synapses or the nerve terminal ultrastructure. Analysis of synaptic function in primary PRRT2 knockout excitatory neurons by live imaging and electrophysiology showed slowdown of the kinetics of exocytosis, weakened spontaneous and evoked synaptic transmission and markedly increased facilitation. Inhibitory neurons showed strengthening of basal synaptic transmission, accompanied by faster depression. At the network level these complex synaptic effects resulted in a state of heightened spontaneous and evoked activity that was associated with increased excitability of excitatory neurons in both PRRT2 knockout primary cultures and acute hippocampal slices. The data indicate the existence of network instability/hyperexcitability as the possible basis of the paroxysmal phenotypes associated with PRRT2 mutations. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: email@example.com.