The type 2 5'-deiodinase (D2) appears to play an important role in maintaining the intracerebral T3 content relatively constant during changes in thyroidal state. Previous studies have demonstrated that the regulation of this enzyme by thyroid hormone and its analogs occurs are posttranslational level. The availability of the rat D2 complementary DNA now allows an assessment of whether pretranslational regulation of this enzyme also occurs in the cerebral cortex. In rats rendered hypothyroid by the addition of methimazole to the drinking water, D2 messenger RNA (mRNA) is increased 70% (P = 0.03). Treatment with L-T3 (50 μg/100 g BW) for 4 days results in an 80% decrease in D2 mRNA compared with that in euthyroid controls (P < 0.001). Administration of lower doses of L-T3 (0.25-3 μg/100 g BW · day) is associated with a dose-dependent decrease in cortical D2 mRNA, but little or no change in D2 activity. The decrease in D2 mRNA in response to T3 treatment can be demonstrated within 4 h. Treatment of hypothyroid rats for 2 weeks with graded doses of L-T4 (0.1-1.5 μg/100 g BW-day) results in a significant decrease in cortical D2 activity, but not mRNA. The association between D2 activity and D2 mRNA in euthyroid, hypothyroid, and hormone- treated rats across a full range of thyroidal states suggests that, L-T4 treatment is associated with greater changes in cortical D2 activity (via posttranslational effects) than mRNA, whereas L-T3 treatment has a greater effect on decreasing D2 mRNA (i.e. pretranslational effects). In conclusion, these studies demonstrate both pre- and posttranslational regulation of cortical D2 expression. The relative contribution of each mechanism depends on the ambient thyroid hormone concentration.