The Cre-loxP recombination system has been used to great advantage in vivo for conditional gene targeting, lineage tracing, and other applications. To express cre in skeletal myoblasts and muscle fibers, we utilized the well-characterized transcriptional regulatory regions of the muscle determination gene, MyoD. Transgenic mouse lines were produced (F3/-2.5cre) in which the cre gene is driven by the MyoD promoter and core enhancer, which directs the early activation of MyoD. Specificity of cre expression and efficiency of recombination was determined by monitoring reporter gene expression after crossing to the Cre-dependent reporter lines, R26R and Z/AP. Efficient labeling of embryonic and fetal myoblasts and muscle fibers was observed, with timing that was similar (branchial arches and limb buds) or slightly delayed (myotomes) relative to the endogenous MyoD gene. In satellite cell cultures, a strict concordance between MyoD protein and reporter gene expression was observed, demonstrating the muscle specificity and efficiency of Cre-mediated recombination. Nascent muscle fibers were labeled following injury of adult muscle, indicating recombination in satellite cells or their daughters in vivo.