The aim of the present study was to evaluate in vivo the selective effects of a small increase in plasma TSH levels on thyroid function, proliferation and morphology. Chronically catheterized male Sprague-Dawley rats were stimulated i.v. over 5 days either with TRH (2 micrograms TRH in 100 microliters 0.9% (w/v) NaCl (TRH-P) or the NaCl carrier alone (P), both given as pulses every 2 h. Control groups were cotreated i.v. with 10 micrograms thyroxine (T4)/100 g body weight per day (TRH-P + T4) starting 2 days before pulsatile stimulation. TSH plasma levels were approximately doubled by TRH-P (P < or = 0.001), T4 plasma levels significantly increased (P < or = 0.001) but tri-iodothyronine plasma levels did not change compared with treatment with P. No significant changes between groups were found in thyroid weight and in intrathyroidal iodine content, but the percentage of 5-bromo-2'-desoxyuridine-labelled thyrocytes as a marker of proliferation in TRH-P-treated animals was significantly increased over P or TRH-P + T4 (P < or = 0.001). Ultrastructural analysis of the thyroid evaluated by electron microscopy revealed a significant increase in the number of lysosomes (P < or = 0.001). The size of the endoplasmic reticulum (ER) in relation to the cytoplasm was significantly increased when treated with TRH-P compared with P or TRH-P + T4 (P < or = 0.001). Post-embedding immunogold staining revealed Tg as a major product within ER cisternae. Immunogold labelling was moderate in controls and higher densities of gold particles were obtained in TRH-P-treated animals (P < or = 0.001). In conclusion, short-term pulsatile TRH stimulation increasing the plasma levels of immunoreactive TSH only twofold is capable of inducing hypertrophy of the thyrocytes by gross ultrastructural changes which are paralleled by an increase in circulating T4. These data underscore the dominant role of TSH on thyroid ultrastructure within the narrow boundaries of normal physiological regulation.