We have cloned and identified a DNA sequence complementary to the mRNA of creatine kinase (CK) isozyme M, although the mRNA is a minor species of the total mRNA in developing myoblasts. Poly(A)+RNA from breast and thigh muscle of 5-week-old chicks was enriched for CK mRNA by a novel procedure of sucrose gradient centrifugation in the presence of methylmercuric hydroxide. DNA complementary to this mRNA was inserted into pBR322, and colonies containing the recombinant plasmids were screened for the ability of the plasmid DNA to hybridize with and rescue CK mRNA from total muscle mRNA. Three plasmids, pCS195, pCS192, and pM35-4, could specifically rescue CK-M mRNA. CK-M mRNA was detected by in vitro translation and specific immunoprecipitation. The identity of the in vitro translation product was further confirmed by its migration in two-dimensional gels at the isoelectric point and molecular weight of CK-M. The heterogeneity of CK-M observed in vivo also was found upon translation of the CK-M mRNA which hybridizes to the plasmid.