Postnatal Sertoli cell maturation is characterized by a pronounced rise in androgen receptor (AR) expression which increases several fold between birth and adulthood. Since both 3,3',5-triiodothyronine (T-3) and FSH regulate Sertoli cell proliferation and differentiation, we :have determined the effects of T-3 and FSH on AR mRNA expression in cultured Sertoli cells from 5-day-old rats. These cultures contain 5-9% peritubular cells, which also express AR mRNA. To insure that the observed T-3 responses did not result from peritubular cells, we examined T-3 effects on AR mRNA expression in cultured 20-day-old Sertoli cells (which contain minimal peri-tubular contamination) and peritubular cells, and measured thyroid hormone receptor (TR) mRNA expression in both of these cell types. Sertoli cells from 5- and 20-day-old rat testes were grown in serum-free medium alone (controls) or with ovine FSH (100 ng/ml) and/or T-3 (100 nM) for 4 days. Peritubular cells purified from 20-day-old rat testes were grown in serum-containing medium for 8 days. These cells were split 1:4, and grown an additional 8 days, the last 4 days in serum-free medium with or without T-3. TR and AR mRNA levels in all cultures were determined by Northern blotting. AR mRNA levels in 5- and 20-day-old cultured Sertoli cells were significantly (P<0.05) increased by both T-3 and FSH alone. Furthermore, AR mRNA levels in Sertoli cells treated with T-3 and FSH were greater than with either alone. TR mRNA expression was detected in cultured peritubular cells, but TR mRNA levels in these cells were only approximately 30% of that seen in 20-day-old cultured Sertoli cells. In contrast to Sertoli cells, T-3 did not affect peritubular AR mRNA expression. These results indicate that T-3 is an important regulator of the postnatal Sertoli cell AR mRNA increase. The additive effects of maximally stimulatory doses of FSH and T-3 suggest these hormones work through different mechanisms to increase AR mRNA. TR mRNA expression in peritubular cells indicates these cells may be direct T-3 targets: though the function of T-3 in these cells is unknown.