The transport of 2-deoxyglucose (dGlc) by cultured rat Sertoli cells was stimulated by l -triiodothyronine (T3) in a time and dose-dependent manner. The lag-time was of about 6 h, the half-maximal dose (ED50) was 0.47 nM, which correlates with the Kd of the nuclear T3 receptor of rat Sertoli cells (Kd = 1–2 nM), and the stimulation was maintained up to 24 h. The effect was specific, as judged by the order of potency of T3 analogs. Cycloheximide prevented the stimulatory effect without affecting the basal uptake. T3 stimulated the uptake of the glucose analog 3-O-methylglucose (MeGlc) with the same order of potency as that of dGlc. The ontogenetic profile of the T3 effect coincides with that of T3 nuclear receptors in rat Sertoli cells. Northern blot analysis demonstrated that Sertoli cells express the erythrocyte/brain glucose transporter isoform (GLUT1) but not the adipose/muscle isoform (GLUT4). T3 treatment (10−7 M for 24 h) induces an increase of GLUT1 mRNA level comparable to that of glucose analog uptake. These results suggest that thyroid hormone stimulates glucose transport by increasing the synthesis of new glucose transporter units and give further evidence for a direct effect of thyroid hormone in the modulation of Sertoli cell functions.