Using RNA interference (RNAi), we specifically down-regulate protein expression in differentiated human skeletal myotube cultures. Serum stimulation of myotubes increases glucose uptake. Using a sensitive photolabeling technique, we demonstrate that this increase in glucose uptake is accompanied by increased cell-surface content of glucose transporter (GLUT) 1. Using RNAi, we specifically reduce GLUT1 mRNA and protein expression, leading to inhibition of serum-mediated increase in glucose transport. Thus, we demonstrate the utility of RNAi in a primary human differentiated cell system, and apply this methodology to demonstrate that serum-mediated increase in glucose transport in human skeletal muscle cells is dependent on GLUT1. (C) 2003 Elsevier Science (USA). All rights reserved.