Dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) are the most abundant steroidal products and major circulating steroids in humans. The serum concentrations of DHEA-S are lower in patients with myotonic dystrophy (DM) than normal controls, and possible improvement of myotonia and muscle weakness was recently reported following DHEA-S replacement therapy. However, the molecular mechanism of action of DHEA-S remains unknown. To understand the reported anti-DM action of DHEA-S, we investigated DHEA-S binding in skeletal muscle cells in vitro. We identified two populations of DHEA-S binding sites (Kd = 5-9 microM and 35-40 microM) in C2C12 myocytes. Similar binding sites were also identified in human skeletal muscles. The Kd value of the high-affinity site was within the range of serum concentrations of DHEA-S in adult humans. Our results suggest that DHEA-S might act directly on skeletal muscles under normal physiological conditions in humans.