Supraoptic nucleus (SON) neurons secrete oxytocin or vasopressin in response to various physiological stimuli (e.g., lactation/suckling, dehydration). Released near fenestrated capillaries of the neurohypophusis, these peptides enter the blood and travel to peripheral target organs. The pervasive neuromodulator adenosine, acting at A₁ receptors, is an important inhibitory regulator of magnocellular neuroendocrine cell activity. Another high-affinity adenosine receptor exists in this system, however. We examined the physiological effects of adenosine A_2A receptor activation and determined its localization amongst various cell types within the SON. In whole-cell patch clamp recordings from rat brain slices, application of the selective adenosine A_2A receptor agonist, CGS 21680, caused membrane depolarizations in SON neurons, often leading to increased firing activity. Membrane potential changes were persistent (>10 min) and could be blocked by the selective A_2A receptor antagonist ZM 241385, or GDP--S, the latter suggesting postsynaptic sites of action. However, MCPG or tetrodotoxin also blocked CGS 21680 effects, indicating secondary actions on postsynaptic neurons. In voltage clamp mode, application of CGS 21680 caused a slight increase (~8%) in high-frequency clusters of excitatory postsynaptic currents. Using specific antibodies, adenosine A_2A receptors were immunocytochemically localized to both the magnocellular neurons and astrocytes of the SON. Ecto-5'nucleotidase, an enzyme involved in the metabolism of ATP to adenosine, was also localized to astrocytes of the SON. These results demonstrate that adenosine acting at A_2A receptors can enhance the excitability of SON neurons and modulate transmitter release from glutamatergic afferents projecting to the nucleus. We suggest that adenosine A_2A receptors may function in neuroendocrine regulation through both direct neuronal mechanisms and via actions involving glia.