Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interferon-γ (INF-γ) have inhibitory effects on thyroid function both in vivo and in vitro. We have studied the effects of these cytokines on type I 5'-deiodinase (5'-DI) mRNA expression and enzyme activity in FRTL-5 cells maintained in standard cell culture medium containing 0 (5H) or 2 mIU/ml bovine TSH (6H). Northern blots were hybridized with 5'-DI cDNA. 5'-DI mRNA levels were reduced to 20% of control values after treating cells with 100 ng/ml TNF-α in 6H for 2 days while the corresponding enzyme activity was reduced 50%. Other cytokines, including IL-1β and interferon-γ, also significantly inhibited expression of 5'-DI in FRTL-5 cells grown in 6H medium. Because the majority of circulating T₃ in the rat is secreted by the thyroid gland, the highly significant decline in the serum T₃/T₄ ratio following in vivo administration of cytokines may be due to their direct inhibitory effect on thyroidal 5'-DI expression.