Acute stimulation of A 3 adenosine receptors has been shown to intensify damage in an in vivo model of global cerebral ischemia and to induce cell death in cell cultures. To investigate the mechanism for these cytotoxic phenomena, adenosine analogs of varied receptor subtype selectivities were applied directly to rat cerebellar granule neurons in culture. Agonists of A 1 , A 2A , and/or A 2B receptors (CPA, NECA and CGS 21680) had no effect on neuronal survival during a 16 h incubation period. The highly selective adenosine A 3 receptor agonist 2-chloro-N 6 -(3-iodobenzyl)adenosine-5´-N-methyluronamide (Cl-IB-MECA) at ≥10 µM induced cell death in a concentration-dependent fashion. A structurally related nucleoside, cladribine, which has cytotoxic properties via a non-adenosine receptor related mechanism, had no effect on cell survival. Adding dbcAMP to the culture to activate cyclic AMP-dependent protein kinases attenuated Cl-IB-MECAinduced neurotoxicity, suggesting the Cl-IB-MECA-induced neuronal cell death is mediated by an inhibition of cyclic AMP production. Glutamate (50 µM) induced cell death to a degree comparable to that induced by 10 µM Cl-IB-MECA. Furthermore, a subcytotoxic concentration of Cl-IB-MECA (1 µM) significantly augmented glutamate neurotoxicity. In contrast, the adenosine A 1 /A 2 agonists, the adenosine A 1 /A 2 antagonist XAC, and the A 2A antagonist SCH 58261 at micromolar concentrations had no effect on neuronal viability or on glutamate neurotoxicity. These results suggest that activation of adenosine A 3 receptors may play an important role in regulating neuronal survival and death in cerebellar neurons.