Spontaneous miniature excitatory postsynaptic currents (MEPSCs) were recorded by whole-cell voltage-clamp techniques in cultured rat hippocampal pyramidal neurons. The specific adenosine A1 receptor agonist cyclopentyladenosine (CPA) reduced the frequency of MEPSCs without affecting their amplitude distribution or kinetic properties. This action was blocked by pretreatment of the cells with pertussis toxin. In the presence of divalent cation Ca2+ channel blockers, CPA was still effective in reducing the frequency of MEPSCs. It was shown that this effect cannot be explained by changes in basal Ca2+ influx. These results suggest that neurotransmitters that produce presynaptic inhibition at hippocampal synapses utilize several mechanisms, one of which may involve inhibition of some component of the quantal release apparatus that occurs independently of inhibition of Ca2+ influx.