The question of whether thyroxine (T4) and TRH have a mitogenic effect on pituitary thyrotrophs and somatotrophs in thyroidectomized rats was investigated. Mitoses were counted in hematoxylin-eosin-stained or periodic acid-Schiff-hematoxylin-stained pituitary slides or immunostained for TSH or GH using male rats thyroidectomized for 5 months. Ten days before they were killed groups of rats were injected with different doses of T4 (0.5, 3 or 10 micrograms i.m. every second day for 10 days), TRH alone (100 ng s.c. three times a day for 10 days), or T4 plus TRH (same doses as above). Mitoses (stopped with colchicine) were counted in 1 mm2 areas at a magnification of x1000. In thyroidectomized rats, mitoses were not significantly increased and treatment with TRH or 0.5 microgram T4 alone in thyroidectomized rats did not affect mitotic counts. In thyroidectomized rats treated with higher doses of T4, mitoses were increased in a dose-dependent fashion. Simultaneous administration of TRH and T4 had a significant synergistic effect on pituitary mitoses in a T4 dose-dependent manner. The treatments also had differential effects on the relative percentages of cellular types in mitosis. Thus, 60% somatotrophs and 12.5% thyrotrophs were found in the euthyroid group. In thyroidectomized and thyroidectomized plus TRH groups, no somatotrophs in mitosis were seen, while thyrotrophs were 28.5% and 33.3% respectively. In thyroidectomized rats treated with low doses of T4, somatotrophs and thyrotrophs in mitosis increased to 38.4% and 80% respectively and, with simultaneous administration of a low dose of T4 plus TRH, although less effective than T4 alone, mitosis increased in somatotrophs and thyrotrophs to 11.1% and 54.5% respectively. A high dose of T4 alone did not increase the mitotic figures in somatotrophs (38.8%), while it diminished the percentage of thyrotrophs to 25%. The administration of high doses of T4 plus TRH had an opposite effect on the mitotic figures of somatotrophs and thyrotrophs and thus the percentage of somatotrophs increased to 50% while thyrotrophs decreased to 5.5%. Ten days of treatment with T4 were insufficient to reverse the histology to euthyroidism. It can be concluded that in long-standing hypothyroidism: (1) thyroid hormone replacement elicits a dose-dependent and differential proliferative response on pituitary thyrotrophs and somatotrophs, (2) TRH is devoid of mitogenic effects when administered alone and (3) the proliferative response of somatotrophs to T4 is enhanced by its co-administration with TRH, suggesting a permissive and/or synergistic effect of the thyroid hormone and TRH.