Basal and FSH-induced aromatase activity in prepubertal rat Sertoli cells was inhibited by L-triiodothyronine (T3) in a time- and dose-dependent manner. The effect was evident only after 6 h of preincubation with T3 (10(-7) M) and the half-maximal dose was 0.5 +/- 0.2 nM, which correlated with the Kd of the nuclear T3 receptor of rat Sertoli cells (Kd = 1-2 nM). The effect was specific as judged by the lack of effect of the T3 analogue 3-iodo-L-tyrosine. The inhibitory effect of T3 was present over the entire range of FSH concentrations used (0.001-100 ng/ml). In T3-treated Sertoli cells, aromatase activity induced by 8-bromo-cyclic AMP was inhibited by the same order of magnitude as that of FSH, thus suggesting that the inhibitory effect of T3 was downstream from cyclic AMP formation. Furthermore, pretreatment of Sertoli cells cultures with T3 (24 h, 10(-7) M) did not affect basal or FSH-induced extracellular cyclic AMP accumulation. This effect of T3 on rat Sertoli cell aromatase activity may be regarded as a part of the integrated mechanism by which thyroid hormone modulates the functions of the seminiferous epithelium.