Excessive muscling in double-muscled cattle arises from mutations in the myostatin gene, but the role of myostatin in normal muscle development is unclear. The aim of this study was to measure the temporal relationship of myostatin and myogenic regulatory factors during muscle development in normal (NM)- and double-muscled (DM) cattle to determine the timing and possible targets of myostatin action in vivo. Myostatin mRNA peaked at the onset of secondary fiber formation (P < 0.001) and was greater in DM (P < 0.001) than in NM. MyoD expression was also elevated throughout primary and secondary fiber formation (P < 0.001) and greater in DM (P < 0.05). Expression of myogenin peaked later than MyoD (P < 0.05); however, it did not differ between NM and DM. These data show that myostatin and MyoD increase coincidentally during formation of muscle fibers, indicating a coordinated role in the terminal differentiation and/or fusion of myoblasts. Myostatin mRNA is also consistently higher in DM than NM, suggesting that a feedback loop of regulation is also disrupted in the myostatin-deficient condition.