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). T₃ regulates the CDKI p27^Kip1 in other cell types, and mice lacking p27^Kip1 have increased testis size. To test the hypothesis that T₃ 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 5-25 days postnatal, 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-day-old Sertoli cells, but had increased strongly in non-proliferating 25-day-old Sertoli cells. p27^Kip1 immunostaining was reduced in Sertoli cells from hypothyroid mice compared with euthyroid controls at 10 and 16 days, 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 10 and 16 days postnatal, but p27^Kip1 expression was comparable by day 25. Hyperthyroidism increased p27^Kip1 immunostaining relative to controls, and Western analysis indicated that Sertoli cells from 10-day-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 T₃ effects on Sertoli cell proliferation may be mediated through this CDKI.