Microglial cells in brain and spinal cord are characterized by high expression of the chemokine receptor CX₃CR1. Expression of the sole CX₃CR1 ligand, the membrane-tethered and sheddable chemokine CX₃CL1/fractalkine, is restricted in the brain parenchyma to selected neurons. Here we summarize our current understanding of the physiological role of CX₃CR1 for microglia function and the CX₃C axis in microglial/neuronal crosstalk in homeostasis and under challenge. Moreover, we will discuss the efforts of our laboratory and others to exploit CX₃CR1 promoter activity for the visualization and genetic manipulation of microglia to probe their functional contributions in the central nerve system (CNS) context.