Studies were undertaken to define the existence of specific T3-binding sites in the synaptosomal fraction of rat cerebral cortex. Development-related changes of the synaptosomal T3-binding sites were compared with those of nuclear T3 receptors in rat cerebral cortex. Scatchard analysis was compatible with the existence of two sets of high affinity T3-binding sites: K1, with a mean (+/- SE) apparent dissociation constant (Kd) of 3.22 +/- 0.63 x 10(-11) M and a mean (+/- SE) maximum binding capacity (MBC) of 3.2 +/- 0.3 pg T3/mg protein, and K2, with a K4 of 2.64 +/- 0.15 x 10(-9) M and a MBC of 218.3 +/0 15.0 pg T3/mg protein. When compared to T3, 27-, 135-, 500-, and 1000-fold higher concentrations of T4, 3,5-diiodothyronine (3,5-T2), triiodothyroacetic acid, and rT3, respectively, were needed to obtain 50% depression of the synaptosomal T3 binding. Tetraiodothyroacetic acid, 3,3'-T2, 3'5'-T2, 3-monoiodothyronine (3-T1), 3'-T1, and thyronine had little effect. The MBC of T3 nuclear receptors was the highest at 2 days of age and significantly decreased thereafter, although an almost constant K4 was observed in all age groups studied. On the contrary, the MBC of the higher affinity synaptosomal T3-binding sites was low in 2-day-old rats (20.3 +/- 1.2 pg T3/g tissue) and increased thereafter to the level in young adult ras (50.4 +/- 2.2 pg T3/g tissue). However, the Kd of synaptosomal T3-binding sites was also unchanged in all age groups studied. These findings clearly indicate that there exist specific T3-binding sites in synaptosomes from rat cerebral cortex.