Loss of skeletal muscle profoundly affects the health and well-being of patients, and there currently is no way to replace lost muscle. We believe that a key step in the development of a prosthesis for reconstruction of dysfunctional muscular tissue is the ability to reconstitute the in vivo-like 3-dimensional (3D) organization of skeletal muscle in vitro with isolated satellite cells. In our present proof of principle studies, we have successfully constructed a multilayered culture of skeletal muscle cells, derived from neonatal satellite cells, that are distributed in a 3D pattern of organization that mimics many of the features of intact tissue. These multilayered cultures are composed of elongated multinucleated myotubes that are MyoD positive. Histological studies indicate that the multiple layers of myotubes can be distinguished. Expression of muscle-specific markers such as myosin heavy chain, dystrophin, integrin alpha-7, alpha-enolase, and beta-enolase was detected using real-time reverse transcriptase polymerase chain reaction at levels near adult values. Physiological measurements of the engineered skeletal muscle showed that they tetanize and display physiologic force length behavior, although developed force per cross-sectional area was below that of native rat skeletal muscle. © Copyright 2007, Mary Ann Liebert, Inc.