Sarcomeric mitochondrial creatine kinase (sMtCK) is one component of a multiprotein, high energy channeling complex consisting of porin, mitochondrial creatine kinase, and adenine nucleotide translocase. To study the transcriptional mechanisms specifying sMtCK gene expression to the heart, transgenic mice were created carrying the 5'-flanking sequences of the human sMtCK gene ligated upstream of the human growth hormone (hGH) reporter gene. RNA blot hybridization demonstrated that the human sMtCK sequence, -485 to +6 base pair (bp), did not activate reporter gene expression to a detectable level. However, the human sMtCK sequence, -921 to +6 bp, expressed the hGH reporter gene at a high level in heart and skeletal muscle and at a very low level in esophagus and kidney, and it did not express the hGH gene in other organs tested (brain, lung, liver, spleen, bladder, uterus, and stomach). In situ hybridization revealed that reporter gene transcription was specified to cardiac and skeletal myocytes, recapitulating precisely the expression pattern of the endogenous gene. Sequence analysis identified several consensus binding sites between -921 and -757 bp, including four GATT motifs, one E box, and one MEF2 site. Further analysis of a third transgenic mouse strain demonstrated that the human sMtCK sequence, -757 to +6 bp, did not direct detectable expression of the hGH reporter gene. We conclude that this 160-bp genomic sequence, from -921 to -757 bp, is necessary in specifying expression of the human sMtCK gene to the oxidative and highly metabolically active heart tissue.