Many lines of evidence indicate a role for thyroid hormones in the expression of cognitive and affective disorders. These conditions constitute a large proportion of the illness burden in the general population. Unfortunately, presently available diagnostic procedures cannot adequately identify these problems. To determine whether imaging studies of thyroid hormone kinetics in brain might be feasible in patients with these disorders, an autoradiographic method for measuring thyroid hormone kinetics was developed. Twenty-five awake adult rats received high specific activity [(125)I]-triiodothyronine (T(3)*). Brains were obtained at intervals from 5 through 300 min after i.v. hormone administration. Every 5th frozen section was thaw mounted and exposed to film. To determine whether T(3) was responsible for the autoradiographic images, the intervening sections were assembled while frozen in regional tissue pools and were extracted and then analyzed by high-performance liquid chromatography. The results demonstrated that radioactivity was almost entirely due to T(3)*( approximately 90%) while small amounts of hormone metabolites, including [(125)I]iodine accounted for the remainder. Regional concentrations of label in autoradiograms were measured by densitometry in hippocampus (CA1, CA2, CA3, and dentate gyrus), cerebellum (molecular and granular cell layers), caudate nucleus, and amygdala. Unexpectedly and interestingly, the results demonstrated that binding through 5 h was mainly irreversible. Regional values of the net uptake rate constant of T(3)* or influx constant, K(i), were determined from the time course of the T(3)* data, showing significant differences among regions. These results suggest that imaging of labeled thyroid hormone ligands by positron emission tomography or single photon emission computed tomography may be feasible and would potentially provide useful information relevant to T(3) processing in the brain during a variety of drug and disease-induced conditions.