Excitatory glutamatergic synapses in the hippocampal CA1 region of rats are potently inhibited by purines, including adenosine, ATP, and ATP analogs. Adenosine receptors are A1 known to mediate at least part of the response to adenine nucleotides, either because adenine nucleotides activate A1 receptors directly, or activate them secondarily upon the nucleotides’ conversion to adenosine. In the present studies, the inhibitory effects of adenosine, ATP, the purportedly stable ATP analog adenosine-5′-O-(3-thio)triphosphate (ATPγS), and cyclic AMP were examined in mice with a null mutation in the adenosine A1 receptor gene. ATPγS displaced the binding of A1-selective ligands to intact brain sections and brain homogenates from adenosine A1 receptor wild-type animals. In homogenates, but not in intact brain sections, this displacement was abolished by adenosine deaminase. In hippocampal slices from wild-type mice, purines abolished synaptic responses, but slices from mice lacking functional A1 receptors showed no synaptic modulation by adenosine, ATP, cAMP, or ATPγS. In slices from heterozygous mice the dose-response curve for both adenosine and ATP was shifted to the right. In all cases, inhibition of synaptic responses by purines could be blocked by prior treatment with the competitive adenosine A1 receptor antagonist 8-cyclopentyltheophylline. Taken together, these results show that even supposedly stable adenine nucleotides are rapidly converted to adenosine at sites close to the A1 receptor, and that inhibition of synaptic transmission by purine nucleotides is mediated exclusively by A1 receptors.