Type II iodothyronine 5'-deiodinase catalyzes the bioactivation of thyroid hormone in the brain. In astrocytes, this approximately 200-kDa, membrane-bound enzyme is composed of at least one p29 subunit, an approximately 60-kDa, cAMP-induced activation protein, and one or more unidentified catalytic subunit(s). Recently, an artificial type II-like selenodeiodinase was engineered by fusing two independent cDNAs together; however, no native type II selenodeiodinase polypeptide is translated in the brain or brown adipose tissue of rats. These data suggest that the native type II 5'-deiodinase in rat brain is unrelated to this artificial selenoprotein. In this report, we describe the cloning of the 29-kDa subunit (p29) of type II 5'-deiodinase from a lambdazapII cDNA library prepared from cAMP-induced astrocytes. The 3.3-kilobase (kb) cDNA encodes an approximately 30-kDa, 277-amino acid long, hydrophobic protein lacking selenocysteine. Northern blot analysis showed that a 3.5-kb p29 mRNA was present in tissues showing type II 5'-deiodinase activity such as brain and cAMP-stimulated astrocytes. Domain-specific, anti-p29 antibodies specifically immunoprecipitated enzyme activity. Overexpression of exogenous p29 or a green fluorescence protein (GFP)-tagged p29 fusion protein led to a >100-fold increase in deiodinating activity in cAMP-stimulated astrocytes, and the increased activity was specifically immunoprecipitated by anti-GFP antibodies. Steady-state reaction kinetics of the enzyme in GFP-tagged p29-expressing astrocytes are identical to those of the native enzyme in brain. Direct injection of replication-deficient Ad5-p29(GFP) virus particles into the cerebral cortex of neonatal rats leads to a approximately 2-fold increase in brain type II 5'-deiodinating activity. These data show 1) that the 3.3-kb p29 cDNA encodes an essential subunit of rat type II iodothyronine 5'-deiodinase and 2) identify the first non-selenocysteine containing subunit of the deiodinase family of enzymes.