1. Electrical responses to volleys in afferent fibers in the optic tract were recorded in the superficial gray layer of anesthetized rat superior colliculus. A prominent negative wave with 4- to 6-ms peak latency in the upper part of the superficial gray layer and a sharp negative wave with 1.5- to 2-ms peak latency in the lower part of the superficial gray layer were elicited, corresponding to the C2 (upper part of the superficial gray layer) and the C1 (lower part of the superficial gray layer) postsynaptic potentials reported by Sefton. 2. These C1 and C2 waves were depressed by kynurenic acid or quinoxaline dione (DNQX) applied just beside the recording electrode, suggesting that neurotransmission in these pathways is mediated by glutamate. 3. Adenosine (10 microM) injected in the superficial gray layer enhanced both C1 and C2 potentials up to 170 and 140%, respectively. 4. Administration of a potent inhibitor of adenosine deaminase, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA; 5 mg/kg sc) increased the amplitudes of both C1 and C2 potentials to 125 and 130% of the initial levels, respectively. 5. The extracellular application of adenosine uptake inhibitors, dipyridamole (100 microM) and nitrobenzylthioinosine (NBI; 10 microM) also enhanced postsynaptic potentials. 6. Prior application of L-homocysteine thiolactone (10 microM), a compound that facilitates the incorporation of adenosine into S-adenosylhomocystein and reduces the extracellular concentration of adenosine, attenuated the excitatory action of exogenously applied adenosine. 7. Excitatory effects were also observed upon application of a selective adenosine A1 receptor agonist, N6-cyclohexyladenosine (CHA) or a selective A2 receptor agonist, 2-[4-(2-carboxylethyl)- phenethylamino]-5'N-ethylcarboxamide adenosine hydrochloride (CGS21680). Selective A1 and A2 receptor antagonists, 8-cyclopentyl-1,3-dimethylxanthine (CPT) and 3,7-dimethyl-1-propargylxanthine (DMPX), respectively, failed to suppress the excitatory action by adenosine. However, combined application of these two agents blocked the facilitatory action by adenosine on the excitatory synapses. 8. The application of adenosine (10 microM) to the superficial gray layer via a microdialysis probe increased the glutamate release by approximately 230% of the basal level. Similarly, the administration of EHNA (5 mg/kg sc) enhanced the extracellular glutamate level up to approximately 170%. However, prior application of L-homocysteine thiolactone (10 microM) failed to potentiate the glutamate release by adenosine. 9. This is the first in vivo study to demonstrate an excitatory action of adenosine on synaptic transmission.(ABSTRACT TRUNCATED AT 250 WORDS)