Spontaneous activity in the central nervous system is strongly suppressed by blockers of gap junctions (GJs), suggesting that GJs contribute to network activity. However, the lack of selective GJ blockers prohibits the determination of their site of action, i.e. neuronal versus glial. Astrocytes are strongly coupled through GJs and have recently been shown to modulate synaptic transmission, yet their role in neuronal network activity was not analysed. The present study investigated the effects and site of action of the GJ blocker, carbenoxolone (CBX), on neuronal network activity. To this end, we used cultures of hippocampal or cortical neurons, plated on astrocytes. In these cultures neurons display spontaneous synchronous activity and GJs are found only in astrocytes. CBX induced in these neurons a reversible suppression of spontaneous action potential discharges, synaptic currents and synchronised calcium oscillations. Moreover, CBX inhibited oscillatory activity induced by the GABAA antagonist, bicuculline. These effects were not due to blockade of astrocytic GJs, since they were not mimicked nor occluded by endothelin-1 (ET-1), a peptide known to block astrocytic GJs. Also, these effects were still present in co-cultures of wild-type neurons plated on astrocytes originating from connexin-43 (Cx43) knockout mice, and in neuronal cultures which contain few isolated astrocytes. CBX was not likely to exert its effect through neuronal GJs either, as immunostaining for major neuronal connexins (Cx) as well as dye or electrical coupling, were not detected in the different models of cultured neurons examined. Finally while CBX (at 100 μm) did not modify presynaptic transmitter release and postsynaptic responses to glutamate, it did cause an increase in the action potential threshold and strongly decreased the firing rate in response to a sustained depolarising current. These data demonstrate that CBX does not exert its action on network activity of cultured neurons through astrocytic GJs and suggest that it has direct effects on neurons, not involving GJs.