We have cloned and characterized the rat GLUT4 gene in order to identify the cis-DNA elements responsible for tissue-specific GLUT4 expression. In this study, a variety of luciferase reporter gene constructs were transiently transfected into C2C12 myoblasts and myotubes as a model for skeletal muscle differentiation. These data identified a 103-base pair fragment, located from -522 to -420 relative to the transcription initiation site, that was sufficient to account for GLUT4 C2C12 myotube-specific expression. This fragment was operationally defined as an enhancer since it conferred myotube-specific expression in the context of both the minimal native GLUT4 or the heterologous thymidine kinase promoters in an orientation-independent manner. Further, mutagenesis of this fragment demonstrated that a sequence analogous to the muscle creatine kinase myocyte enhancer factor 2 (MEF2) binding site (-466 and -457) was required for transcriptional activation. Electrophoretic mobility gel shift assays demonstrated specific binding activity to the GLUT4 MEF2 sequences which directly correlated with functional expression. Although this element was capable of directing myotube-specific expression when cloned as multiple copies into luciferase reporter gene constructs, the MEF2 sequence alone was insufficient to enhance GLUT4 expression. These data demonstrated that GLUT4 muscle-specific expression is conferred by a 103-base pair DNA sequence located between -522 and -420 of rat GLUT4 gene. This region encompasses a MEF2 binding site which was necessary, but not sufficient, for transcriptional activation.