In the present study we investigated the biochemical properties of in vitro phenolic (5'D) and tyrosyl (5D) ring deiodination and the tissue concentrations of T4, T3, and rT3 in adult human central nervous system (CNS) tissue. All samples were obtained from nontumoral tissue at autopsy (n = 6) or neurosurgical operation (n = 5). Both phenolic and tyrosyl ring deiodinase activities were demonstrable in all samples obtained intraoperatively, whereas only tyrosyl ring deiodination was evident in the tissues obtained postmortem. The phenolic ring deiodination pathway corresponded to the type II 5'-deiodinase isoenzyme with regard to its high affinity for T4 and rT3 (Km = 2.2 and 2.4 nmol/L, respectively), its insensitivity to 6-propyl-n-2-thiouracil (PTU), and the sequential reaction mechanism. No PTU-sensitive 5'-deiodination of rT3 was demonstrable. Tyrosyl ring deiodination of both T4 and T3 showed typical type III 5D kinetics (Ka, 6.5 nmol/L for T4 and 3.4 nmol/L for T3) and was PTU insensitive. Nanomolar concentrations of tissue T4, T3, and rT3 were detected in samples obtained both intraoperatively and postmortem. They were very similar to the absolute values of the apparent Km for T4, T3, and rT3 in the phenolic and tyrosyl ring deiodination pathways. In conclusion, we have demonstrated the coexistence of both phenolic and tyrosyl ring deiodinase activities in the human CNS. Their kinetic characteristics, substrate specificity, and reaction mechanisms are very similar to the corresponding type II 5'- and type III 5-iodothyronine deiodinase activities in rat brain. In contrast to the findings in the rat CNS, no PTU-sensitive phenolic ring deiodinase (i.e. type I 5'D) activity was found in the human CNS. This may explain the relatively high tissue concentrations of rT3.