A significant retention of [125I]triiodothyronine ([125I]T3) in the retrobulbar orbital area of mice has been previously shown. The present study was initiated to determine tissue and intracellular localization of the thyroid hormone in the above area which is concerned in human Graves' disease of the thyroid. Male and female Balb C mice were intravenously injected with 0.1 mL of [125I]T3 (0.2 mCi/micrograms). At various time intervals (30 s-10 min) the animals were sacrificed, bled and periorbital tissues were isolated under a dissecting microscope. Three series of samples were prepared: (a) frozen samples for cryomicrotome sections, (b) samples fixed in 10% formaldehyde for paraffin embedded tissues and (c) samples fixed in paraformaldehyde (2%), glutaraldehyde (2%) and 0.1 M sodium cacodylate for embedding in Epon-Araldite-DDSA. Sections 5 microns and 400-600 A thick for light and electron microscopy, respectively, were coated with Ilford L4 emulsion and exposed for 9-21 days. Light microscope autoradiography demonstrated that [125I]T3 injected intravenously is rapidly transported in the cells of fat tissue of the peribulbar orbital area and tissues with glandular or muscular function: the hormone showed a high affinity for the intra- and extraorbital lacrymal gland cells, the cells of the Harder's gland, those of the sebaceous and meibomian glands of the eye-lids, as well as for local muscular structures. Electron microscope autoradiography showed that radioactivity is already localized inside the cells 30 s after the i.v. injection of [125I]T3 and it is distributed throughout the cytoplasm, with a higher concentration in the vesicles of the Harder's gland cells (rich in lipids and porphyrin), in the endoplasmic reticulum and the mitochondria of the lacrymal glands. 10 min after injection, a shifting of the radioactivity towards the nucleus area was observed. In conclusion, after in vivo injection, the thyroid hormone rapidly penetrates the cells of fat glandular and muscular tissues in the orbital area. Intracellularly, the affinity of the hormone for the secretory vesicles, rough endoplasmic reticulum, mitochondria and nucleus suggest that T3 could play a role in secretory and metabolic functions of the tissues in the retrobulbar orbital area.