The effects of thyroid hormone on the levels of protein inhibitors for Ca2+-dependent proteinase were studied in rat brain. Four different inhibitory proteins (I, II, III, and IV) were isolated from immature (7-day-old) rat brain. The molecular weights of these inhibitory proteins, which were estimated by gel-exclusion chromatography on Sephacryl S-200 column, were approximately 280,000 (I), 70,000 (II), 50,000 (III), and 35,000 (IV). All of these inhibitory proteins were decreased by thyroidectomy. Four-day T4 administration (100 micrograms/kg daily) to thyroidectomized-immature animals restored proteins I, II, and IV. However, protein III was not recovered with the same treatment of the rats. In mature rats (40-day-old), four different inhibitory proteins were identified in the brain. The molecular weights were identical to those obtained in immature rat brain. As observed in immature rats, all of these inhibitory proteins were decreased by thyroidectomy. In contrast to the results obtained in immature animals, however, inhibitory protein III, as well as I, II, and IV, was restored by T4 administration (100 micrograms/kg daily) to thyroidectomized mature rats. The results suggested that thyroid hormone increases protein inhibitors for Ca2+-dependent proteinase in brain, and that irreversible decrease in one of the inhibitors is induced by thyroid hormone deficiency in immature animals. The phenomenon may be related to the neonatal hypothyroidism-induced irreversible damage to the central nervous system in patients with cretinism.