The activity of type I 5'-deiodinase (5'DI) is known to correlate with thyroid status; it is high in hyperthyroidism and low in hypothyroidism. Recently, it was shown that the increased activity of type I 5'DI in hyperthyroidism is associated with an increase in enzyme contents as well as its mRNA in liver. However, it remains unknown whether thyroid hormone directly regulates the expression of 5'DI mRNA in hepatocytes. In this study, the direct actions of thyroid hormone as well as rT3 and dexamethasone on type I 5'DI mRNA were investigated using primary cultures of rat hepatocytes. Hepatocytes were prepared from euthyroid rats by collagenase perfusion and plated either on collagen-coated dishes for conventional monolayer cultures or on positively charged dishes for spheroid cultures. After hormonal treatments, the levels of mRNAs for type I 5'DI and albumin were determined by Northern blotting. In spheroid cultures, T3 increased type I 5'DI mRNA in a dose- and time-dependent manner, whereas the albumin mRNA level was not altered. A lesser effect was observed in hepatocytes cultured as monolayers. The T3-induced increase in 5'DI mRNA was not inhibited by pretreatment with cycloheximide, indicating that the effect of thyroid hormone on 5'DI mRNA is direct, not requiring de novo protein synthesis. rT3 did not affect the levels in type I 5'DI mRNA increased by T3. On the other hand, dexamethasone alone increased 5'DI mRNA and, when added together with T3, had a synergistic effect. In contrast to T3, dexamethasone increased albumin mRNA. Dexamethasone-induced increases in mRNAs for 5'DI and albumin were inhibited by pretreatment of cycloheximide. The present study indicated that T3 increases 5'DI mRNA through a direct action on its gene, whereas the effect of dexamethasone requires de novo synthesis of a protein factor(s).