Expression of the muscle creatine kinase (MCK) gene in skeletal and heart muscle is controlled in part by a 5' tissue-specific enhancer. In order to identify new regulatory elements, we designed mutations in a previously untested conserved portion of this enhancer. Transfection analysis of these mutations delineated a new control element, named Trex (Transcriptional regulatory element x), which is required for full transcriptional activity of the MCK enhancer in skeletal but not cardiac muscle cells. Gel mobility shift assays demonstrate that myocyte, myoblast, and fibroblast nuclear extracts but not primary cardiomyocyte nuclear extracts contain a trans-acting factor that binds specifically to Trex. The Trex sequence is similar (7/8 bases) to the TEF-1 consensus DNA-binding site involved in regulating other muscle genes. To determine if TEF-1 interacts with Trex, selected TEF-1 binding sites such as GTIIc and M-CAT and two anti-TEF-1 antisera were used in gel shift assays. These experiments strongly suggest that a factor distinct from TEF-1 binds specifically to Trex. Thus it appears that MCK transcription is regulated in skeletal muscles through a Trex-dependent pathway while Trex is not required for MCK expression in heart. This distinction could account partially for the difference in levels of muscle creatine kinase in these tissues.