Inactivating mutations of the mammalian myostatin gene are associated with increased muscle mass and decreased fat mass; conversely, myostatin transgenic mice that overexpress myostatin in the skeletal muscle have decreased muscle mass and increased fat mass. We investigated the effects of recombinant myostatin protein and antimyostatin antibody on myogenic and adipogenic differentiation of mesenchymal multipotent cells. Accordingly, 10T(1/2) cells were incubated with 5'-azacytidine for 3 d to induce differentiation and then treated with a recombinant protein for myostatin (Mst) carboxy terminal 113 amino acids or a polyclonal anti-Mst antibody for 3, 7, and 14 d. Cells were also cotransfected with a Mst cDNA plasmid expressing the full-length 375-amino acid protein (pcDNA-Mst375) and the silencer RNAs for either Mst (pSil-Mst) or a random sequence (pSil-RS) for 3 or 7 d, and Mst expression was determined. Adipogenesis was evaluated by quantitative image analysis of fat cells before and after oil-red-O staining, immunocytochemistry of adiponectin, and Western blot for CCAAT/enhancer binding protein-alpha. Myogenesis was estimated by quantitative image analysis-immunocytochemistry for MyoD (Myo differentiation protein), myogenin, and myosin heavy chain type II, or by Western blot for myogenin. 5'-Azacytidine-mediated differentiation induced endogenous full-length Mst expression. Recombinant Mst carboxy terminal 113 amino acids inhibited both early and late markers of myogenesis and stimulated both early and late markers of adipogenesis, whereas the antibody against Mst exerted the reverse effects. Myogenin levels at 7 d after transfection of pcDNA-Mst375 were reduced as expected and elevated by pSil-Mst, which blocked efficiently Mst375 expression. In conclusion, myostatin promotes the differentiation of multipotent mesenchymal cells into the adipogenic lineage and inhibits myogenesis.