Type 2 deiodinase (D2) and type 3 deiodinase (D3) locally achieve the determination of the concentration of T₃, which binds to the thyroid hormone receptor with high affinity. D2 converts T₄ into T₃, and D3 degrades T₄ and T₃. Neurons take up T₃ released by astrocytes, the main cerebral site for the D2 expression. Because oxidative stress is believed to be involved in several neurological disorders, we explored the effects of oxidative stress on D3 and D2 in primary culture of rat astrocytes. H₂O₂ (250 μm) increased D3 activity with maximal effects around 8 h. Stimulation of D3 activity by H₂O₂ was synergistic with T₄, phorbol ester, and also cAMP. H₂O₂ (250 μm) did not affect basal D2 activity but inhibited the stimulation of D2 activity by cAMP and factors implicating cAMP-independent pathways in astrocytes, TSH, and phorbol ester. N-Acetyl cysteine and selenium repletion, which respectively increase intracellular glutathione and glutathione peroxidase, inhibited D2 and D3 regulation by H₂O₂, whereas l-buthionine sulfoximine, which decreases intracellular glutathione, mimicked H₂O₂ effects. Oxidative stress up-regulated D3 and inhibited cAMP-stimulated D2 by transcriptional mechanisms. A decrease in cAMP by oxidative stress could contribute to the inhibition of cAMP-stimulated D2. Using specific inhibitors of signaling pathways, we show that the ERK pathway was required in D2 and D3 regulation by oxidative stress and that the p38 MAPK pathway was implicated in H₂O₂-induced D3. We suggest that the expected decrease in T₃ might modulate the cellular injury of oxidative stress in some pathological brain conditions.