Possible myogenic differentiation of SSEA-1- and OCT-4-positive murine embryonic stem cells (ESCs) and embryoid bodies (EBs) was studied in vitro and in vivo. In vitro, ESC- or EB-derived ESCs (EBs/ESCs) showed only traces of Pax 3 and 7 expression by immunocytochemistry and Pax 3 expression by immunoblot. By RT-PCR, myogenic determinant molecules (myf5, myoD, and myogenin) were expressed by EBs/ESCs but not by ESCs. However, in such cultures, very rare contracting myotubes were still present. Suspensions of LacZ-labeled ESCs or EBs were injected into anterior tibialis muscles (ATM) of different cohorts of mice for the study of their survival and possible myogenic differentiation. The different cohorts of mice included isogenic adult 129/Sv, nonisogenic CD1 and mdx, as well as mdx immunosuppressed with 2.5 mg/kg daily injections of tacrolimus. Ten to 90 days postinjections, the injected ATM of nonisogenic mice did not contain cells positive for LacZ, SSEA-1, OCT-4, or embryonic myosin heavy chain. The ATM of intact mdx mice contained very rare examples of muscle fibers positive for dystrophin and/or embryonic myosin heavy chain. In the ATM of the isogenic normal and the immunosuppressed mdx mice, as expected, large teratomas developed containing the usual diverse cell types. In some teratomas of immunosuppressed mdx mice, small pockets of muscle fibers expressed dystrophin and myosin heavy chain. Our studies indicated that in muscles of animals nonisogenic with the used ESCs, only very rare ESCs survived with myogenic differentiation. These studies also indicated that ESCs will not undergo significant, selective, and preferential myogenic differentiation in vitro or in vivo in any of the models studied. It is probable that this strain of murine ESC requires some experimentally induced alteration of its gene expression profile to secure significant myogenicity and suppress tumorogenicity.