The discovery of tetrahydrogestrinone (THG) abuse by several elite athletes led the U.S. Congress to declare it a controlled substance, although conclusive evidence of its anabolic/androgenic activity is lacking. We determined whether THG affects myogenic differentiation and androgen receptor (AR)-mediated signaling, whether it binds to AR, and whether it has androgenic and anabolic effects in vivo. Accordingly, we measured the dissociation constant for THG with a fluorescence anisotropy assay using recombinant AR-ligand binding domain. The AR nuclear translocation and myogenic activity of androstenedione were evaluated in mesenchymal, multipotent C3H10T1/2 cells. We performed molecular modeling of the THG:AR interaction. The androgenic/anabolic activity was evaluated in orchidectomized rats. THG bound to AR with an affinity similar to that of dihydrotestosterone. In multipotent C3H10T1/2 cells, THG upregulated AR expression, induced AR nuclear translocation, dose dependently increased the area of myosin heavy chain type II-positive myotubes, and up-regulated myogenic determination and myosin heavy chain type II protein expression. The interaction between AR and the A ring of THG was similar to that between AR and the A ring of dihydrotestosterone, but the C17 and C18 substituents in THG had a unique stabilizing interaction with AR. THG administration prevented the castration-induced atrophy of levator ani, prostate gland, and seminal vesicles and loss of fat-free mass in orchidectomized rats. We conclude that THG is an anabolic steroid that binds to AR, activates AR-mediated signaling, promotes myogenesis in mesenchymal multipotent cells, and has anabolic and androgenic activity in vivo. This mechanism-based approach should be useful for rapid screening of anabolic/androgenic agents.