Ciliary neurotrophic factor (CNTF) is a potent survival molecule for a large number of neuronal and glial cells in culture; its expression in glial cells is strongly upregulated after a variety of nerve tissue injuries. Exogenously administered CNTF produces an anorectic effect via activation of hypothalamic neurons and stimulates neurogenesis in mouse hypothalamus. To determine whether CNTF is produced endogenously in the hypothalamus, we sought cellular sources and examined their distribution in adult mouse hypothalamus by immunohistochemistry. CNTF immunoreactivity (IR) was predominantly detected in the ependymal layer throughout the rostrocaudal extension of the third ventricle, where numerous ependymocytes and tanycytes exhibited specific staining. Some astrocytes in the grey matter of the anterior hypothalamus and in the median eminence of the hypothalamic tuberal region were also positive. Stimulation of cells bearing CNTF receptor α (CNTFRα) induces specific activation of the signal transducer and activator of transcription 3 (STAT3) signalling system. Treatment with recombinant CNTF and detection of the nuclear expression of phospho-STAT3 (P-STAT3) showed that CNTF-producing ependymal cells and tanycytes were intermingled with, or very close to, P-STAT3-positive, CNTFRα-bearing cells. A fraction of CNTF-producing ependymal cells and tanycytes and some median eminence astrocytes also exhibited P-STAT3 IR. Thus, in normal adult mice the ependyma of the third ventricle is both a source of and a target for CNTF, which may play hitherto unknown roles in hypothalamic function in physiological conditions.