The stimulation of glucose transport by 3,5,3'-triiodo-L-thyronine (T3) in the liver-derived ARL 15 cell line is only partly attributable to increased GLUT-1 glucose transporter gene expression. To test the hypothesis that T3 increases the partitioning of GLUT-1 to the cell surface, we quantitated surface GLUT-1 using the photolabel ATB-[3H]BMPA. In control cells only approximately 20% of total cellular GLUT-1 was present at the cell surface. T3 treatment (100 nM) for 6 h increased the rate of 2-deoxy-[3H]glucose (2-DG) uptake by 30, 92, and 95% in three experiments and increased surface GLUT-1 photolabeling by 17, 81, and 72%, respectively, with no increase in total cellular GLUT-1. T3 treatment for 48 h increased 2-DG uptake by 143, 172, and 216% in three experiments and increased cell surface GLUT-1 photolabeling by 88, 161, and 184%, respectively, with smaller increases in total cellular GLUT-1. T3 treatment for 48 h thus increased the fraction of cellular GLUT-1 at the plasma membrane from 21 +/- 2 to 35 +/- 3% (SE). We conclude that most of the early (6-h) stimulation of glucose transport by T3 in ARL 15 cells is mediated by an increase in the partitioning of GLUT-1 to the plasma membrane. With more chronic T3 treatment (48 h), the enhanced surface partitioning of GLUT-1 is persistent and is superimposed on an increase in total cellular GLUT-1, accounting for a further increase in glucose transport.