Circulating dehydroepiandrosterone (DHEA) is converted to testosterone in the target tissues. Recently, we demonstrated that skeletal muscles are capable of locally synthesizing circulating DHEA to testosterone and estrogen. Furthermore, testosterone is converted to 5-dihydrotestosterone (DHT) by 5-reductase, and exerts biophysiological actions through binding to androgen receptors. However, it remains unclear whether skeletal muscle can synthesize DHT from testosterone and/or DHEA and whether these hormones affect glucose metabolism-related signalling pathway in skeletal muscles. We hypothesized that locally synthesized DHT from testosterone and/or DHEA activates glucose transporter-4 (GLUT4) -regulating pathway in skeletal muscles. The aim of the present study was to clarify whether DHT is synthesized from testosterone and/or DHEA in cultured skeletal muscle cells and whether these hormones affect the GLUT4-related signalling pathway in skeletal muscles. In the present study, the expression of 5-reductase mRNA was detected in rat cultured skeletal muscle cells and the addition of testosterone or DHEA increased intramuscular DHT concentrations. Addition of testosterone or DHEA increased GLUT4 protein expression and its translocation. Furthermore, Akt and protein kinase C / (PKC/) phosphorylations, which are critical in GLUT4-regulated signalling pathways, were enhanced by testosterone or DHEA addition. Testosterone- and DHEA-induced increases in both GLUT4 expression and Akt and PKC/ phosphorylations were blocked by a DHT inhibitor. Finally, the activities of phosphofruktokinase and hexokinase, main glycolytic enzymes, were enhanced by testosterone or DHEA addition. These findings suggest that skeletal muscle is capable of synthesizing DHT from testosterone, and that DHT activates the glucose metabolism-related signalling pathway in skeletal muscle cells.