We investigated the role of the A 3 adenosine receptor in cells of the astroglial lineage (both rat primary astrocytes and human astrocytoma ADF cells) by means of the selective A 3 agonists N 6 -(3iodobenzyl)-adenosine-5ยข-N-methyluronamide (IB-MECA) and CI-IB-MECA, and by utilizing the selective A 3 receptor antagonist MRS1191. Exposure of ADF cells to mM concentrations of either agonist resulted in reduction of cell number, likely due to cell death. In both rat astrocytes and human astrocytoma cells, at concentrations 2-3 orders of magnitude lower (which were not associated with cytotoxicity), these same agonists induced a marked reorganization of the cytoskeleton, with appearance of stress fibers and numerous cell protrusions. Functionally, these morphological changes were associated with cell protection, as demonstrated by a significant reduction of spontaneous apoptosis in A 3 agonist-treated cells. To confirm a role for the A 3 receptor in this effect, MRS1191 completely counteracted CI-IB-MECA-induced reduction of spontaneous apoptosis. In ADF cells, A 3 agonists also induced changes in the intracellular distribution of the anti-apoptotic protein Bcl-X L , which became localized in cell protrusions. Also, this effect was specifically antagonized by MRS1191. These dual actions of A 3 agonists in vitro may have important in vivo implications. For example, a robust and acute activation of the A 3 receptor following massive adenosine release during ischemia may contribute to brain cell death; conversely, a subthreshold activation of this receptor prior to ischemia may trigger protective mechanisms (i.e., induction of stress fibers and of a Bcl-X Ldependent reorganization of cytoskeleton) making the brain more resistant to subsequent insults ("ischemic tolerance").