The expression and functions of kainate-type glutamate receptors (KARs) in the hippocampus are developmentally regulated. In particular, presynaptic KARs depressing glutamate release are tonically activated during early postnatal development, and this activity is down-regulated in parallel with maturation of the synaptic circuitry. In order to understand the physiological relevance of the tonic KAR-mediated signalling, we have here studied the effect of long-term pharmacological activation of KARs on glutamatergic synaptic connectivity in hippocampal slice cultures where presynaptic KARs are expressed but not endogenously activated. Prolonged (16-20 h) activation of the GluR5 subunit-containing KARs using the agonist ATPA (1 microM) caused a specific and enduring increase in the number of glutamatergic synapses in area CA1, evidenced as an increase in the frequency of action potential-independent spontaneous EPSCs (mEPSCs) and in immunostaining against synaptic marker proteins. The long-term ATPA treatment had no detectable effect on GABAergic transmission or on glutamate release probability. Further, the effect of ATPA on synaptic density was independent of action potential firing and dependent on protein kinase C. A critical role of endogenous KAR activity in synaptic development was revealed by chronic treatment of the cultures with the selective GluR5 antagonist LY382884, which caused a significant impairment of glutamatergic transmission to CA1 pyramidal neurons. Together, these data suggest a role for the GluR5 subunit-containing KARs in the formation and/or stabilization of functional glutamatergic synapses in area CA1.