Spontaneous excitatory postsynaptic currents (sEPSC) were recorded in neocortical rat pyramidal neurones in vitro using conventional whole-cell voltage clamp technique, in the presence of tetrodotoxin (TTX: 10 μM) and picrotoxin (100 μM). Experiments were carried out on transverse 280-300 μm thick slices of CBL57 mice (15- to 45-day-old) brain. The animals were humanly killed. Application of glutamate receptors antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX: 50 μM) and D(-)-2-amino-5-phosphonopentanoic acid (D-APV: 30 μM) led to a significant but not complete reduction of sEPSC amplitude. The residual sEPSCs (rsEPSCs) were not affected by combined application of (2S,4R)-4-methylglutamic acid (SYM2081: 10 μM, to desensitize kainate receptors) and (RS)-4-(4-aminophenyl)-1,2-dihydro-1- methyl-2-propylcarbamoyl-6,7-methylenedioxyphthalazine (SYM2206: 50 μM, a non-competitive AMPA receptor antagonist). This suggests that rsEPSCs are mediated neither by AMPA/kainate nor by NMDA receptors. The amplitude of the rsEPSC was 67 ± 15% (mean ± SD, n = 41) of the control sEPSC, and the frequency of residual synaptic events was 39 ± 20% (n = 41) of control. The rsEPSCs were not affected by the nicotinic receptors antagonist hexamethonium (100 μM) or the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX: 1 μM). They were inhibited by P2X receptors antagonists pyridoxal- phosphate-6-azophenyl-2′,4′-disulfonate (PPADS:10 μM) and 8,8′-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid (NF279: 1 μM). The amplitude distribution of synaptic currents in control conditions showed two distinct peaks. The major peak with higher amplitude disappeared after application of CNQX. The minor peak was not altered by glutamate receptor antagonists, but was shifted towards lower amplitudes by PPADS and suramin. Although there was no large difference in the kinetics of sEPSC before and after CNQX, a difference was revealed by cyclothiazide (CTZ). Application of CTZ (50 μM) did not alter the kinetics of the synaptic current recorded in CNQX (decay time 10 ± 2 ms, n=8), whereas the decay time of current recorded in the presence of suramin (10 μM) increased more than two-fold (decay time 19 ± 3 ms, n=5). The distribution of decay times of the sEPSC was unimodal in control conditions, but became bimodal in CTZ with the major peak increased two-fold. The above results suggest the existence of distinct population of purinergic synaptic events which occur asynchronously with respect to those mediated by glutamate. Although the probability of unitary ATP-mediated synaptic events is much lower, their amplitude is comparable to the amplitude of glutamate-mediated events. This may imply a specific role for the purinergic component of excitatory synaptic transmission