Purinergic receptors play a key role in neuron-glia and glia-neuron interactions. In the present study, we have recorded cytosolic Ca²⁺ responses using confocal imaging in astrocytes of acute olfactory bulb slices from mice (postnatal days 3–8). By application of agonists and antagonists, we identified two types of receptors, P2Y₁ and A_2A, that mediated Ca²⁺ responses attributable to Ca²⁺ release from intracellular stores in the astrocytes. Both receptor types were activated by application of ATP and ADP; however, when enzymatic ATP degradation was suppressed by the alkaline phosphatase inhibitor levamisole, ATP only activated MRS2179-sensitive P2Y₁ but not ZM241385-sensitive A_2A receptors. The dose-response curve for A_2A receptors activated by adenosine revealed an EC₅₀ of 0.3 µM, one order of magnitude smaller than the EC₅₀ of 5 µM determined for P2Y₁ receptors activated by ADP. Electrical stimulation of the olfactory nerve in the presence of glutamate receptor blockers to suppress excitation of postsynaptic neurons evoked Ca²⁺ responses in most of the astrocytes, which were inhibited by blocking both P2Y₁ and A_2A receptors. Our results indicate that olfactory nerve terminals release not only glutamate, but also ATP, which activates P2Y₁ receptors and, after degradation of ATP to adenosine, A_2A receptors in astrocytes.—Doengi, M., Deitmer, J. W., Lohr, C. New evidence for purinergic signaling in the olfactory bulb: A_2A and P2Y₁ receptors mediate intracellular calcium release in astrocytes.