Genetically modified myogenic cells have a number of potentially relevant applications for gene therapy of genetic defects. Retroviral vectors proved to be a safe and efficient tool to transfer and express genes into satellite cells and their differentiated progeny, although muscle-specific regulation of the transferred gene is very difficult to achieve in a conventional vector framework. We modified a Moloney murine leukemia virus (MoMLV)-derived retroviral vector containing a bacterial beta-galactosidase (beta-Gal) reporter gene by inserting a muscle creatinine kinase (MCK) enhancer element into the U3 region of the viral long terminal repeat (LTR). The resulting vector (mLBSN) was transferred into cells of different histological origin, including undifferentiated murine and human myogenic cells, which were unable to express the transgene at detectable levels. Instead, gene expression from the modified LTR was obtained in a mouse myogenic cell line and in human primary satellite cells upon induction of differentiation into myotubes in culture, and correlated with the activation of the muscle differentiation program. beta-Gal-negative, mLBSN-transduced human satellite cells were also transplanted into the quadricep muscle of immunodeficient mice, where activation of the transgene expression was observed in vivo after differentiation and fusion into muscle fibers. These results show that retroviral vectors carrying LTRs modified in the enhancer sequences may be used to target tissue- and differentiation-specific gene expression into the muscle. For practical purposes, satellite cells engineered by muscle-specific retroviral vectors might represent an effective tool to deliver expression of a given gene product specifically into the muscle tissue, avoiding undesired protein accumulation in mononucleated cells. More generally, this type of vector might be useful whenever regulated expression of a transferred gene is necessary in a target cell or tissue.