Fractalkine/CX3CL1 is a neuron-associated chemokine, which modulates microglia-induced neurotoxicity activating the specific and unique receptor CX3CR1. CX3CL1/CX3CR1 interaction modulates the release of cytokines from microglia, reducing the level of tumor necrosis factor-α (TNF-α), interleukin-1β (IL1-β) and nitric oxide and induces the production of neurotrophic substances, both in vivo and in vitro. We have recently shown that blocking adenosine A1 receptors (A1R) with the specific antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) abolishes CX3CL1-mediated rescue of neuronal excitotoxic death and that CX3CL1 induces the release of adenosine from microglia. In this paper we demonstrate that the presence of extracellular adenosine is mandatory for the neurotrophic effect of CX3CL1 since reducing adenosine levels in hippocampal cultures, by adenosine deaminase (ADA) treatment, strongly impairs CX3CL1-mediated neuroprotection. Furthermore, we confirm the predominant role of microglia in mediating the neuronal effects of CX3CL1, since the selective depletion of microglia from hippocampal cultures treated with clodronate-filled liposomes causes the complete loss of effect of CX3CL1. We also demonstrate that hippocampal neurons obtained from AR mice are not protected by CX3CL1 whereas AR neurons are. The requirement of functional AR for neuroprotection is not unique for CX3CL1 since AR hippocampal neurons are not rescued from Glu-induced cell death by other neurotrophins like brain-derived neurotrophic factor (BDNF) and erythropoietin (EPO), which are fully active on wt neurons.