In the central nervous system, the active form of thyroid hormone, T3, derives from the cellular uptake and intracellular 5′-monodeiodination of T4 by type II 5′-monodeiodinase (DII). Here, we report that using an antiserum raised against the C-terminus of the full-length SeDII, immunolabeled cells were found in the rat hypothalamus in agreement with the DII mRNA distribution. Light and electron microscopy shows that DII is localized in astrocytes and tanycytes, supporting the hypothesis that these cells play an important role in the mediation peripheral signals, such as thyroid hormones, on hypothalamic functions.