Transient neonatal hypothyroidism in the rat causes prolonged Sertoli cell proliferation, delayed Sertoli cell maturation, and increased adult Sertoli cell number, testis weight, and sperm production. Conversely, neonatal hyperthyroidism decreases Sertoli cell proliferation and ultimate testis size. This suggests that thyroid hormones might normally directly inhibit Sertoli cell proliferation while promoting maturation. However, these Sertoli cell effects could be due to secondary hormonal or metabolic effects of hypo- or hyperthyroidism. In this study, we directly tested thyroid hormone effects on Sertoli cell proliferation and differentiation in vitro. Sertoli cells from 5-day-old rat testes were grown in serum-free medium alone (controls) or with additional triiodothyronine (T₃; 1–200 nM) and/or FSH (1 µg/ml). After 4 days, cultures were used to obtain RNA for Northern hybridization or for thymidine autoradiography. Labeling index (L) for control cultures and cultures receiving 100 nM T₃ alone was 5.2 ± 0.5 and 5.0 ± 0.4, respectively. The LI of FSH-treated cultures increased to 8.4 ± 0.8 (p < 0.01 vs. control). Cultures treated with FSH and 1, 10, 100, or 200 nM T3 had LIs of 8.0 ± 0.9, 6.1 ± 0.4, 5.3 ± 0.6, and 4.8 ± 0.6, respectively; the last three values were less than for cells receiving FSH alone (p < 0.01) or FSH + 1 nM T3 (p < 0.05). Northern hybridization indicated that mRNA levels for clusterin and inhibin-β_B, Sertoli cell secretory proteins whose production normally increases during postnatal differentiation in vivo, were significantly increased by T₃ or FSH alone. Furthermore, mRNA levels for inhibin-β_B in Sertoli cells treated with both T₃ and FSH were greater than with either alone, indicating that these hormones can act synergistically to promote maturation. These results indicate that T₃ directly decreases mitogenesis in FSH-stimulated Sertoli cells and stimulates production of mRNA for secretory proteins characteristic of the more mature cell. Thus, T₃ may normally directly promote differentiation of Sertoli cells from the mitotic to nonmitotic state and the concomitant onset of secretory function. These results also suggest that the changes in Sertoli cell mitogenesis and differentiation, and eventual changes in adult testis size and sperm production, following neonatal hypo- or hyperthyroidism may result from direct effects of the altered levels of thyroid hormone on Sertoli cells during neonatal development.