Interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) have many effects on a number of cell types, including thyrotrophs. In the present study, we used FRTL5 cells, a cultured rat thyroid follicular cell line, to examine the effects of IFN-gamma and TNF-alpha on type I 5'-deiodinase (5'D-I) activity and 5'D-I, thyroid peroxidase (TPO) and thyroglobulin (Tg) gene expression. Incubation of FRTL5 cells with the highest concentrations of TNF-alpha and IFN-gamma tested (1000 ng/ml or 1000 U/ml, respectively) for 72 h in the presence and absence of TSH had no effect on cell viability as assessed by trypan blue exclusion. In TSH-deprived FRTL-5 cells, TNF-alpha and IFN-gamma resulted in a small but dose-dependent decrease in 5'D-I activity. TNF-alpha or IFN-gamma blocked the TSH- or cAMP-induced rise in 5'D-I activity. 100 ng/ml TNF-alpha and 100 U/ml IFN-gamma completely blocked the TSH- or cAMP-induced rise in 5' D-I activity. However, when cells were incubated with TNF-alpha and IFN-gamma, in combination, there was a marked decrease in 5'D-I activity, with TNF-alpha (25 ng/ml) plus IFN-gamma (25 U/ml) completely blocking the TSH-induced rise in 5'D-I activity. Northern blot analyses were performed to examine the effect of TNF-alpha and IFN-gamma on 5'D-I gene expression. TNF-alpha had little effect on 5'D-I messenger RNA (mRNA) levels, while IFN-gamma resulted in a modest decrease in 5'D-I mRNA levels in TSH-deprived cells, and in TSH-stimulated FRTL-5 cells. However, when TNF-alpha and IFN-gamma were added in combination there was a marked decrease in 5'D-I gene expression with TNF-alpha (50 ng/ml) plus IFN-gamma (50 U/ml) decreasing 5'D-I mRNA levels by 89 percent in TSH-deprived cells. In TSH-stimulated cells incubated with 500 ng/ml TNF-alpha plus 500 U/ml IFN-gamma, 5'D-I mRNA levels were almost undetectable. We also examined the effect of IFN-gamma and TNF-alpha on TPO and Tg gene expression. As observed with 5'D-I mRNA levels, there was a synergistic effect of IFN-gamma and TNF-alpha on the inhibition of basal and TSH-stimulated TPO and Tg gene expression. These findings indicate that TNF-alpha and IFN-gamma in combination have a marked inhibitory effect on thyroid function, which is consistent with a decrease in thyroid hormone synthesis and metabolism.