We recently reported that tumor necrosis factor-α (TNF-α) induction of the synthesis and secretion of transforming growth factor (TGF)-β1 by FRTL-5 cells is a thyroid-stimulating hormone (TSH)-dependent and age-dependent process⁽⁹⁾ TNF-α is only cytotoxic to aged (>40 passages) FRTL-5 cells grown in TSH-containing medium, whereas TGF-β induces programmed cell death (apoptosis) in epithelial cells but not in FRTL-5 cells, which otherwise retain many properties of normal thyroid follicular cells. This cell line is, therefore, a convenient model for studies on the TSH-dependent and age-dependent inhibitory effects of these cytokines on epithelial cell growth, viability, and function. One prominent effect of TNF-α (and TGF-β1) on FRTL-5 cell function is suppression of iodide uptake, which is markedly stimulated by TSH. In aged FRTL-5 cells, iodide uptake is only about 10% that of young control cells. Na⁺/K⁺-ATPase activity, which drives iodide uptake by thyroid cells, is inhibited by TNF-α and TGF-β. The following experiments quantitate the effects of TSH, aging, TNF-α, and TGF-β1 on the expression and activity of Na⁺/K⁺-ATPase activity in FRTL-5 cells. Young (<20 passages) and aged (>40 passages) FRTL-5 cells were treated with various doses (0–100 ng/ml) of recombinant human TNF-α or TGF-β1 for various times (0–3 days) with and without 2 U/liter TSH. These treatments reduced the rate-limiting Na⁺/K⁺-ATPase βl mRNA level and Na⁺/K⁺-ATPase activity in parallel in a dose-dependent and time-dependent fashion. Aged FRTL-5 cells were more sensitive to the inhibitory effects of TNF-α, whereas young cells were more sensitive to the suppressive effects of TGF-βl on the expression and activity of Na⁺/K⁺-ATPase. We conclude that inhibition of Na⁺/K⁺-ATPase activity by TNF-α and TGF-β in FRTL-5 cells is differentially affected by aging and that this inhibitory effect can be dissociated from effects on cell viability.