Cells of the murine skeletal muscle line, C2C12, undergo differentiation from mononuclear myoblasts to multinuclear myotubes that express a number of proteins associated with striated muscle. We examined the relationship between the abundance of the mRNAs encoding the fast-twitch Ca-ATPase and the alpha isoforms of Na,K-ATPase and the subsequent expression of their respective polypeptides. Both the mRNA and protein levels of the alpha 1 isoform remained constant throughout differentiation. In contrast, the content of mRNAs encoding the alpha 2 isoform and fast-twitch Ca-ATPase increased coordinately with the abundance of their corresponding polypeptides during myotube development. Despite the dramatic increase in alpha 2 expression, estimates of in vitro Na,K-ATPase activity and assessments of in vivo transport activity suggest that alpha 2 contributes little to ionic homeostasis in C2C12 myotubes.