Procalcitonin (PCT) is a 116-amino acid polypeptide physiologically produced, as the precursor protein of calcitonin (CT), in the parafollicular cells of the thyroid gland, but physiological functions and other major sources of PCT remains unclear. The distribution of PCT-like immunoreactivity (PCT-LI) in the rat hypothalamus was examined by immunohistochemistry using a monoclonal antibody raised against the mid-region of human PCT (60-77-amino acid fragment). This antibody cross-reacts well with rat PCT and immature CT, but it cross-react poorly with free mature CT. Abundant expression of PCT-LI was found in zones at the interface between brain and cerebrospinal fluid (CSF) such as the ependymal layer and ventral glia limitans (VGL). Double labeling of PCT and glial fibrillary acidic protein (GFAP) identified this population of small cells as astrocytes, possibly tanycytes, a type of specialized glial cell that interacts in neuroendocrine functional dynamics. The fibers of these cells extend to circumventricular organs (CVOs) and to astrocytes located inside the parenchyma of key autonomic regulatory hypothalamic areas, with highest densities in the supraoptic nucleus (SO), arcuate nucleus (Arc), area postrema (AP), median eminence (ME), medial preoptic nucleus, tuber cinereum, and accessory neurosecretory nuclei. No strongly labeled cells were found in the paraventricular nucleus. The wide distribution of PCT-LI in the hypothalamus, in close correspondence with previous mapping of CT receptors in the rat brain, suggests that PCT may influence a multitude of biological activities associated with the hypothalamic-pituitary axis.