1. We studied adenosine's action on synaptic transmission from primary afferent fibers to neurons of the substantia gelatinosa (SG) using tight-seal whole cell recordings in transverse slices of hamster spinal cord. Adenosine had two actions, hyperpolarization of the postsynaptic membrane and depression of the excitatory postsynaptic currents (EPSCs) evoked by dorsal root stimulation. 2. Under voltage clamp adenosine elicited a sustained outward current at a holding potential of -70 mV. The outward current was blocked by a combination of intracellular cesium and tetraethylammonium, an effect characteristic of potassium channels. The adenosine-induced current reversed at -97 +/- 6 (SD) mV, close to the potassium equilibrium potential. These observations suggest that adenosine activates a potassium conductance in SG neurons so as to inhibit primary afferent synaptic transmission postsynaptically. 3. Adenosine reduced the miniature EPSC frequency without significantly changing the amplitude. In contrast, the glutamate receptor competitive antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) substantially reduced the amplitudes of miniature EPSCs while producing a much smaller effect on the miniature frequency than adenosine. In evoked EPSCs adenosine reduced unitary content without reducing unitary amplitude. The effects on both miniature and evoked EPSCs suggest that adenosine inhibits synaptic currents by suppressing presynaptic transmitter release. 4. EPSCs evoked by dorsal root stimuli were subdivided into monosynaptic and polysynaptic categories. Adenosine at superfusion concentrations of 20-300 microM suppressed all polysynaptic EPSCs. Less than half of monosynaptic EPSCs were inhibited, usually those evoked by the slowest-conducting primary afferents. These observations were interpreted to indicate that a principal action of adenosine in SG is on interneuronal communication.