To determine whether mitogen-regulated expression of skeletal muscle genes is independent of cell type, muscle and nonmuscle cells were transfected with cloned 5'-flanking sequences of muscle creatine kinase (MCK) fused to a heterologous reporter gene and tested for expression in high and low mitogen culture conditions. Consistent with the behavior of endogenous MCK, a -3300MCK-CAT gene is expressed at high levels in differentiated muscle cells but at low to undetectable levels in proliferating myoblasts and in either mitogen-deprived or stimulated nonmuscle cells of mesodermal, ectodermal, or endodermal origin. A -776MCK-CAT gene behaves similarly with respect to its cell type specificity but it supports only an intermediate expression level in response to mitogen deprivation in skeletal muscle cells. These data suggest that the -3300 to +7 nucleotide region of mouse MCK contains one or more elements which are activable by mitogen deprivation only in myogenic cells.