Thyroid hormone regulates early postnatal Sertoli cell proliferation. Transient neonatal hypothyroidism allows prolonged postnatal Sertoli cell mitogenesis and doubles adult Sertoli cell numbers, testis weight, and sperm production. The mechanism of this effect is unknown. Cell proliferation is stimulated by cyclins and cyclin-dependent kinases and inhibited by cyclin-dependent kinase inhibitors (CDKIs). T(3) regulates the CDKI p27(Kip1) in other cell types, and mice lacking p27(Kip1) have increased testis size. To test the hypothesis that T(3) regulates Sertoli cell mitogenesis by acting through p27(Kip1), we compared expression of p27(Kip1) in Sertoli cells of testes from euthyroid, hypothyroid, and hyperthyroid mice. At postnatal d 5-25, testes were collected and immunostained for p27(Kip1) expression, or Sertoli cells were isolated enzymatically and used for p27(Kip1) Western blotting. p27(Kip1) immunostaining was low in rapidly proliferating 5-d-old Sertoli cells but had increased strongly in nonproliferating 25-d-old Sertoli cells. p27(Kip1) immunostaining was reduced in Sertoli cells from hypothyroid mice compared with euthyroid controls at 10 and 16 d, consistent with increased Sertoli cell proliferation in these mice. Western blotting corroborated the p27(Kip1) immunostaining, and p27(Kip1) expression was greater in Sertoli cells from control compared with hypothyroid mice at postnatal d 10 and 16, but p27(Kip1) expression was comparable by d 25. Hyperthyroidism increased p27(Kip1) immunostaining relative to controls, and Western analysis indicated that Sertoli cells from 10-d-old hyperthyroid mice expressed more p27(Kip1) than control mice. These results indicate that thyroid hormone status affects p27(Kip1) expression in neonatal Sertoli cells, suggesting that T(3) effects on Sertoli cell proliferation may be mediated through this CDKI.