The regulation of creatine kinase (CK) induction during muscle differentiation was analyzed with MM14 mouse myoblasts. These cells withdraw from the cell cycle and commit to terminal differentiation when fed with mitogen-depleted medium. Myoblasts contained trace amounts of an isozyme of brain CK (designated BB-CK), but differentiation was accompanied by the induction of two other isozymes of muscle and brain CKs (designated MM-CK and MB-CK). Increased CK activity was detectable within 6 h of mitogen removal, 3 h after the first cells committed to differentiation and 6 h before fusion began. By 48 h, MM-CK activity increased more than 400-fold, MB-CK activity increased more than 150-fold, and BB-CK activity increased more than 10-fold. Antibodies prepared against purified mouse MM-CK cross-reacted with muscle and brain CKs (designated M-CK and B-CK, respectively) from a variety of species and were used to demonstrate that the increase in enzymatic activity was paralleled by an increase in the protein itself. CK antibodies were also used to aid in identifying cDNA clones to M-CK. cDNA sequences which corresponded to protein-coding regions cross-hybridized with B-CK mRNA; however, a subclone containing the 3′-nontranslated region was unique and was used to quantitate M-CK mRNA levels during myoblast differentiation. M-CK mRNA was not detectable in myoblasts, but within 5 to 6 h of mitogen withdrawal (6 to 7 h before fusion begins) it accumulated to about 30 molecules per cell. By 24 h, myotubes contained approximately 1,100 molecules per nucleus of M-CK mRNA.