The effect of Tri-iodothyronine (T3) administration leading to the precocius differentiation of Sertoli cell in prepuberal rats has been previously shown. The functional maturation of Sertoli cells is associated with changes in androgen metabolism. We have recently demonstrated that T3 influences androgen metabolism in Sertoli cells by inhibiting aromatase activity and reduces drastically the ER contents in peripubertal hypothyroid rats. To better understand the role of T3 in modulating steroid action on Sertoli cells, we performed a time course study evaluating the in vitro effects of T3 and testosterone (T) on androgen (ARs) and estrogen (ERs) receptor content in Sertoli cells isolated from two weeks old Wistar rats. ARs and ERs basal levels did not change during the time course study indicating that the exposure to culture medium per se did not affect either receptor type. After 24 hrs of incubation with either T3 or T, a decrease of ERs in both nucleus and cytosol was observed. Such a decrease was augmented by the simultaneous administration of both hormones. ARs displayed a different temporal pattern in the two cellular compartments and exhibited an earlier rise in the cytosol induced by either T3 or T. At 36 hrs, ARs were significantly enhanced in both compartments in response to either T or T3 exposure while combined hormonal treatment caused an additive increase compared with the single treatment group. As a consequence of the opposite behaviour pattern displayed by ARs and ERs, the ratio between total ARs and ERs contents was increased after 24 hrs of exposure to hormonal treatment. To evaluate if treatments performed induced a functional maturation of Sertoli cells, transferrin levels in culture medium were measured. The increase of this protein paralleled that of ARs content as well as that of ARs/ERs ratio. This study demonstrates that thyroid hormone induces a progressive increase of (AR)/(ER) ratio in the differentiating Sertoli cells bringing them to a prevalent androgen dependency along their functional maturation.