Sequences complementary to muscle poly(A)+RNA were cloned in the plasmid pBR322 and the resulting colonies were screened by colony hybridization with labeled cDNA derived from skeletal muscle and smooth muscle (gizzard). The skeletal muscle-specific clones were further screened by RNA blotting hybridization for a muscle mRNA having the size expected for a putative type M creatine kinase (M-CK) mRNA. The remaining clones with the expected hybridization properties were finally characterized by hybrid-selected translation, and a cloned sequence was shown to contain DNA hybridizing to mRNA that could be translated into M-CK. This plasmid, pMCK1, was further characterized by restriction mapping. Blot analysis of total cell RNA from differentiating myogenic cell cultures showed accumulation of M-CK mRNA in cultures older than 42 hr but not in young little-differentiated cultures.