The molecular pathways and regulatory molecules that underlie changes in gene transcription during mechanical overload of skeletal muscle remain obscure. To better understand this process, we have examined mouse muscle creatine kinase (MCK) gene expression in mechanically overloaded plantaris (OP) muscle of transgenic and nontransgenic mice. Northern blot analysis revealed that endogenous MCK-specific mRNA transcripts were decreased 150% in the OP muscles after 6 wk. To identify the MCK gene regions involved in the response to mechanical overload, three different mouse MCKCAT transgenes were studied by measuring chloramphenicol acetyltransferase (CAT assays) activity in OP and sham-operated (control plantaris) muscles. Mouse lines carrying (+enh206)117MCKCAT and -1256MCKCAT transgenes exhibited 30 and 40% lower CAT levels, whereas two mouse lines carrying -3300MCKCAT transgenes exhibited average decreases of 430%. Nearly identical results, including measurements of exogenous CAT mRNA, were obtained 2 days postoverload. Six weeks or 2 days of mechanical overload led to an average decrease in MM-CK isoprotein of 140%. These data provide evidence that mechanical overload induces changes in MCK gene expression that appear to be regulated by at least two portions of the MCK gene: the 206 base pair 5' enhancer and the -3,300 to -1,257 region.