Few chemokines are expressed constitutively in the brain at detectable levels; amongst them is fractalkine. We analyzed the distribution of fractalkine in the mouse brain with the aim of giving a neuroanatomical support to the study of its physiological function. To this end, we carried out an analysis of fractalkine protein localization and gene expression. An anti-fractalkine antibody was produced and used to perform an immunohistochemical study. The results indicated a high level of fractalkine protein in cortex, hippocampus, basal ganglia, and olfactory bulb. In particular, the presence of abundant immunoreactive neurons was observed in layers II, III, V, and VI of the cortex. In the hippocampus, the CA1 region was the most intensely labeled, but immunoreactive neurons were present also in CA2 and CA3, whereas in the basal ganglia, immunoreactive cells were observed in the caudate putamen. Other brain structures such as the brainstem showed a few scattered immunoreactive cells. The presence of fractalkine immunoreactive fibers was revealed only in the olfactory bulb and in the anterior olfactory nuclei. Gene expression study results, obtained by both semiquantitative PCR and in situ hybridization, matched protein localization with the highest levels of fractalkine transcript detected in the hippocampus, cortex, and striatum. The present study showed that fractalkine protein and mRNA are constitutively expressed at a high level in forebrain structure, but are almost absent in the hindbrain. Furthermore, localization at the cellular body level would suggest a paracrine or cell-to-cell interaction role for fractalkine more than a neurotransmission modulatory function.