The TSH receptor plays a pivotal role in thyroid gland function, growth, and differentiation, but little is known about its role or regulation in the fetus and neonate. To explore these questions, we systematically evaluated TSH receptor gene expression at the level of messenger RNA (mRNA) in thyroid glands obtained from rat fetuses and neonates, from 14 days gestation to day 5 of postnatal life. Results were compared with histological evidence of differentiation and to thyroid-specific gene expression. Northern blot and RT-PCR analysis revealed that TSH mRNA was first detected at low levels on fetal day 15, but it increased 3- to 15-fold on fetal days 17-18. Up-regulation of TSH receptor mRNA on fetal day 17-18 was accompanied by the first appearance of colloid formation and of follicular development on morphological examination. It was also paralleled by increased expression of the thyroid-specific genes thyroglobulin (Tg) and thyroid peroxidase. Unexpectedly, TSH mRNA abundance was 2- to 3-fold higher in pregnant dams than in nonpregnant adult females or adult males. In view of the 8-day lapse between the first appearance of the thyroid diverticulum and up-regulation of TSH receptor gene expression, we conclude that pituitary TSH, acting through its receptor, plays an important role in terminal thyroid maturation, but it is not involved earlier in gestation. Similarly, these data support previous evidence that the weak thyrotropic activity of human CG could not be of significance in early fetal thyroid gland development. The increased TSH receptor mRNA on fetal day 17-18 may be attributable to up-regulation by TSH, which is first secreted into the fetal circulation at this time. The significance of the increased TSH receptor expression during pregnancy remains to be explored.