Mutations in the myostatin gene lead to double-muscling in cattle indicating that it is a negative regulator of the total number of muscle fibres. Myostatin expression was analysed by RT-PCR in three developing bovine muscles. It decreased during differentiation in Semitendinosus and Biceps femoris, and increased in the late differentiating Masseter during gestation. A combination of in situ hybridisation and immuno-histochemical detection of myosin heavy chains (MHC) allowed us to locate the expression in myofibres containing only developmental MHC at different stages and in fast IIA fibres at the end of gestation. In vitro, myostatin was undetectable during proliferation, peaked at the onset of fusion and decreased during terminal differentiation. It was not detected in myotubes by in situ hybridisation. The inhibition of differentiation by BrdU prevented the decrease in expression. Our results show that the peak in myostatin expression coincides with early differentiation indicating a regulatory role in cattle myogenesis.