Fractalkine/CX3CL1 is a neuron-associated chemokine which modulates microglia-induced neurotoxicity activating the specific and unique receptor CX3CR1. CX3CL1/CX3CR1 pair interaction modulates the release of cytokines from microglia, reducing the level of tumor necrosis factor-a (TNF-a), interleukin-1b (IL1-b) and nitric oxide and inducing the production of neurotrophic substances, both in in vivo and in vitro models. We have recently shown that blocking adenosine receptor type 1 (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 hippocampal neurons obtained from A1R-/- mice are not protected by CX3CL1 which is nevertheless effective on A2AR-/- and A3R-/- neurons. The requirement of functional A1R for neuroprotection is not unique for CX3CL1 since A1R-/- 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 functioning on wt neurons. We also demonstrate that the presence of extracellular adenosine is mandatory for the neurotrophic effect of CX3CL1 since reducing adenosine levels in hippocampal cultures, making use of adenosine deaminase (ADA), 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. Similarly, we demonstrate that CX3CL1 cannot protect cortical neurons from Glu-induced cell death while cortical neurons co-cultured with CX3CL1-stimulated glia are rescued and this effect is strongly reduced by clodronate liposome treatment.