We have studied regulation of the glucose transporter by thyroid hormone in ARL 15 cells, a thyroid hormone-responsive cell line derived from rat liver. T3treatment (5 X 10-8M for 48 h) of confluent cell monolayers grown in thyroid hormone-deficient medium increased the rate of uptake of [3H] 2-deoxyglucose by 2.3 ± 0.2-fold; this effect was half-maximal at a T3concentration of 5 nM. The uptake of the nonmetabol-izable hexose [3H]3-0-methylglucose was comparably increased, confirming a stimulation of glucose transport by thyroid hormone in these cells. In addition to enhancing glucose transporter activity, T3increased the utilization of medium glucose to a similar degree. To elucidate the mechanism of the stimulation of glucose transport by T3, the number of glucose transporter units in crude membrane preparations was quantitated by measuring the glu-cose-inhibitable binding of [3H]cytochalasin-B. The Kdfor specific (glucose-inhibitable) binding of [3H]cytochalasin-B was 5060 nM, a value typical for nonhepatic glucose transporters. T3 treatment caused an increase in the glucose-inhibitable binding of this ligand that was similar in magnitude to the stimulation of [3H]2-deoxyglucose uptake (2.5 ± 0.6-fold). Northern blot analysis of total cellular RNA using a cDNA probe for the rat brain glucose transporter showed a strong 2.9-kilobase hybridization signal after stringent washing, indicating that ARL 15 cells express the specific mRNA for this type of glucose transporter. T3treatment increased the abundance of this mRNA by 2.3 ± 0.2-fold. It is concluded that thyroid hormone stimulates glucose transport in ARL 15 cells, which express the brain type of glucose transporter. This effect is attributable at least in part, if not entirely, to an increase in the level of glucose transporter mRNA and an accompanying increase in the number of glucose transporter units. These findings suggest that thyroid hormone may be an important regulator of glucose transporter gene expression.