In regenerating muscle cells, muscle regulatory factor (MRF) 4 is normally the last of the four MRFs to be expressed. To analyze how the timing of MRF4 expression affects muscle regeneration, we compared regeneration after local freeze injury of muscles from wild-type mice with muscles from transgenic mice in which MRF4 expression was under control of an approximately 1.6-kb fragment of the myogenin promoter. Three days after injury, masseter and tibialis anterior (TA) muscles in wild-type mice expressed little or no MRF4 mRNA; whereas these muscles in transgenic mice expressed abundant MRF4 mRNA from both the transgene and the endogenous gene. Thus, MRF4 up-regulation was accelerated in transgenic compared to wild-type regenerating muscles, and expression of the transgene appeared to activate, perhaps indirectly, expression of the endogenous MRF4 gene. At 11 days after injury, regeneration, as measured by cross-sectional area and density of regenerated fibers, was significantly impaired in transgenic TA compared to wild-type TA, whereas at 19 days after injury both transgenic and TA muscle fibers had fully recovered to preinjury values. Regeneration of masseter muscles, which normally regenerate much less completely than TA muscles, was unaffected by the transgene. Thus, the timing of MRF4 up-regulation, as well as additional muscle-specific factors, can determine the progress of muscle regeneration.