The in vivo internalization of thyrotropin-releasing hormone (TRH) was studied by using a semiquantitative immunoelectron microscopic method. Pituitary glands of normal male rats intravenously injected with 100 ng TRH and sacrificed after 5-60 min were used. Ultrathin sections were obtained by cryoultramicrotomy of fixed pituitary glands. Pituitary cellular types were identified by appropriate antiserums. An antiserum specifically directed against TRH was used. TRH-like immunoreactivity due to endogenous TRH was observed in thyrotropes and prolactin cells, but never in somatotropes, gonadotropes or corticotropes. At the subcellular level, the reaction was detected within the cytoplasmic matrix, the secretory granules, and the nucleus but only occasionally at the plasma membrane. After in vivo injection of TRH, the immunocytochemical reaction was still restricted to thyrotropes and prolactin cells, increased with time elapsed after injection up to 15-30 min and then returned to basal intensity in cytoplasm, secretory granules, and nucleus, and became very frequent at the plasma membrane. These data provide evidence for endogenous TRH within thyrotropes and prolactin cells, i.e., in physiological target cells for TRH, and support the hypothesis that normal TRH target cells can, in vivo, internalize exogenous as well as endogenous TRH into several subcellular compartments including the nucleus.