Thyroid hormone (T3) and insulin are both shown to stimulate glucose transport in Clone 9 cells, a rat liver cell line in which the utilization of glucose is limited by transport rate and in which only the GLUT-1 transporter isoform is expressed. Pre-treatment of these cells with T3 moreover substantially enhances the stimulatory effect of insulin such that at maximally effective hormone concentrations the effects of T3 and insulin on glucose transport are more than additive and indeed nearly multiplicative, suggesting that the mechanisms mediating the enhancement of glucose transport differ between the two hormones. Cell surface biotinylation followed by Western-blot analysis of plasma membrane fractions showed that the stimulatory effects of T3 and insulin on glucose transport, whether acting singly or in combination, exceed the attendant increases in the abundance of GLUT-1 in the plasma membrane. It is suggested that activation of GLUT-1 molecules pre-existing in the plasma membrane plays a major role in mediating the stimulatory effects of T3 and insulin on glucose transport in this cell line.