Jejunal brush-border membrane vesicles were harvested from 4-week old chicks whose thyroid status had been altered either by a daily injection of 20 micrograms T3 for 1 week or which through the preceding 4 weeks had received propylthiouracil and that had been repleted with either 20 or 80 micrograms T3 in divided doses within 48 h. T3 markedly stimulated D-glucose uptake in brush-border membrane vesicles in the presence of an outside/inside (100/0 mmol/l) Na+ gradient. T3 administration had no detectable influence on the Na+ permeability of the isolated vesicles. The effect of the thyroid hormone on Na+ gradient-driven D-glucose uptake was fully preserved at zero transmembrane potential difference. These findings exclude that T3 stimulates Na+-dependent D-glucose transport in the small intestine through changes of the electrochemical Na+ gradient or through alteration of the transmembrane potential difference. Tracer exchange experiments under equilibrium and voltage-clamp conditions revealed a significantly shorter half-time of D-glucose uptake in brush-border membrane vesicles from T3-treated chicks. Kinetic analysis showed that T3 administration significantly increases the apparent maximal velocity of D-glucose transport in brush-border membrane vesicles, whereas the apparent Km values were virtually unaltered. From these data we conclude that T3 increases the activity of Na+-dependent D-glucose carriers in the brush-border membrane. This is interpreted as consistent with a greater rate of D-glucose absorption from the intestinal lumen under conditions of hyperthyroidism.