Recent studies have demonstrated that granule cells in rat cerebellar slices exhibit a tonic form of GABAergic inhibition. The presence of a similar constitutive GABAergic conductance was investigated in synaptically coupled cultures of neonatal rat cerebellum. In cells exhibiting spontaneous inhibitory postsynaptic currents (IPSCs), application of the GABA(A) receptor antagonist bicuculline (10 microM) eliminated the IPSCs and also produced a significant decrease in holding current. This latter effect was lacking in cells that did not exhibit IPSCs. Application of TTX (1 microM) and Cd(2+) (100 microM) decreased the IPSC frequency and also produced a change in holding current; these effects were eliminated by the prior application of bicuculline. In the presence of TTX, application of the benzodiazepine (BDZ) Flunitrazepam (1 microM) caused a 85+/-15% increase in the component of holding current that arose from GABA(A) receptor activity. Noise analysis indicated that the GABA(A) receptors underlying this tonic form of GABAergic inhibition exhibited a mean single channel conductance close to 14 pS, a value similar to that seen for somatic GABA(A) receptors in these cells. Thus, like their counterparts in cerebellar slices, cerebellar granule cells in culture exhibit a background GABAergic conductance. The most likely source of this tonic current is GABA spilt over from active inhibitory synapses. As this conductance was sensitive to benzodiazepine receptor agonists it is unlikely to arise entirely from GABA(A) receptors containing the alpha6 subunit.