Estrogen and thyroid hormones play important roles in the regulation of pituitary function. We presently show that pituitary weight and total cellular RNA levels were significantly decreased by ovariectomy in female rats and were significantly increased by castration in males, without alterations in pituitary DNA levels as compared to intact animals. Treatment with a single dose of estrogen produced a significant increase in pituitary RNA in ovariectomized females but not castrated males. This effect was more obvious following multiple doses of estrogen, and was blocked by pretreatment with cycloheximide, or surprisingly by concomitant administration of triiodothyronine (T3). Analysis of estrogen response element (ERE) binding activity in pituitary nuclear protein extracts revealed that estrogen produced a rapid induction of a slow mobility complex of ERE binding in ovariectomized females much greater than in castrated males. Thus, estrogen-induced increases in pituitary total RNA levels are dependent on new protein synthesis, are gender-specific, are inhibited by T3, and may be mediated via specific estrogen-induced changes in protein-DNA interactions.