T he contribution of L-thyroxine (T4) to nuclear thyroid receptor occupancy was studied in GC cells incubated with concentrations of 3,5,3'-triiodo-L-thyronine (T3) and T4 that resulted in free iodothyronine levels similar to those in serum of euthyroid rats. T4 accounted for 5.4-10% of the occupied receptors: T3 derived from T4 [T3(T4)] and T3 added to medium accounted for the remainder of receptor occupancy. Incubation with increasing medium free T4 resulted in a progressive increase in the contribution of T4 and T3(T4) to receptor occupancy. In incubations with 3.6-fold increased medium free T4, T4 accounted for 20.4%, and T3(T4) for 40.3% of receptor occupancy. These occupancy data and the experimentally determined Ka of thyroid receptor for T3 and T4 allowed calculation of nuclear free iodothyronine concentrations. Nuclear free T3 was 3-6-fold greater than medium free T3 and nuclear [corrected] free T4 was 12-19-fold greater than medium free T4. When GC cells were incubated with decreased medium free T3 and physiological medium free T4, both nuclear receptor occupancy and growth hormone production decreased as well. However, a twofold increase in medium free T4, in the presence of decreased medium free T3, restored receptor occupancy and growth hormone production to or near control values. These findings establish a role for T4 in addition to T3(T4) in nuclear receptor occupancy and biological activity in rat anterior pituitary tissue both in physiologic conditions and when medium free T4 is raised. The findings may have relevance to the sick euthyroid thyroid syndrome in which free T4 may be increased in some patients who have decreased serum free T3.