The relationship between thyroid activity and Sertoli cell function has been investigated in prepubertal rats. Male 28-day-old Wistar rats were used to prepare Sertoli cells by sequential enzyme digestion of the testes. Hypothyroidism, induced by oral administration of methimazole from the day of birth, was characterized by a severe retardation of body and testis growth and a net inhibition of the increase in Sertoli cell gamma-glutamyl transpeptidase (GGT) activity as well as in androgen-binding protein (ABP) and lactate production, which normally occur during postnatal development of Sertoli cells. The functional parameters of Sertoli cells from hypothyroid 28-day-old rats approximated to those of cells from euthyroid 15-day-old animals. These results are consistent with the impairment of protein synthesis in Sertoli cells from hypothyroid rats compared with controls. Body and testis growth were improved and Sertoli cell functions were restored with 3,3',5-tri-iodothyronine (T3) replacement therapy. An excess of T3 in the serum, induced by daily i.p. injections of T3 (100 micrograms/kg body wt) during the last week before the rats were killed, failed to induce changes in body and testis growth or in the activity of GGT and lactate dehydrogenase of Sertoli cells. Cells from hyperthyroid rats exhibited a specific decrease in ABP production. These results indicate that thyroid hormone is necessary for the postnatal maturation of Sertoli cell function and suggest a regulatory role of the hormone on gametogenic development in the prepubertal rat.