The cilated ependymocytes that line the ventricles are decorated by a network of serotoninergic supra-ependymal axons, which are thought to regulate their function. The neurones of origin contain both serotonin and phosphate-activated glutaminase, which raises the possibility that the supra-ependymal axons are also glutamatergic. Using immunocytochemistry, the present study has demonstrated the presence of glutamate in many supra-ependymal axons, as well as in the cilia of ependymocytes. We suggest that glutamate in supra-ependymal axons, counterbalances or opposes the action elicited by serotonin. Glutamate taken up by ependymocytes may supplement metabolic pathways in these cells and could be used to fuel the high energy demands of their cilia.