Skeletal muscle is an attractive target tissue for gene therapy involving both muscle and nonmuscle disorders. HIV-1-based vectors transduce mature skeletal muscle; however, the use of these vectors for human gene therapy may be limited by biosafety concerns. In this study, we investigated gene transfer using lentivirus vectors based on the equine infectious anemia virus (EIAV) in skeletal muscle in vitro and in vivo. EIAV vectors transduce proliferating and differentiating C2C12 mouse muscle cells; furthermore, the addition of the woodchuck hepatitis posttranscriptional element to EIAV vectors markedly increases gene expression in these cells. A single injection of EIAV vectors into skeletal muscle of adult mice led to detectable gene marking and gene expression for the duration of the 3-month study. Use of a second-generation EIAV self-inactivating vector (E-SIN) increased transduction in muscle cells in vitro, and injection of E-SIN vectors into skeletal muscle resulted in increased gene marking and gene expression compared to first-generation EIAV vectors.