This study examined the developmental expression of Na,K-ATPase alpha- and beta-subunit isoforms in different skeletal muscles of the mouse, and the relationship of Na,K-ATPase alpha(2) isoform expression to the developing transverse tubules (t-tubules). We measured Na,K-ATPase and dihydropyridine receptor (DHPR) mRNA and protein in the diaphragm and hindlimb muscles from embryonic day 18.5 (E18.5) to 6 weeks postnatal, using DHPR expression to mark the timing of t-tubule formation. The Na,K-ATPase subunits showed developmental age-dependent and muscle-specific expression that was controlled by both transcriptional and post-transcriptional mechanisms. The alpha(1) isoform is expressed at more constant levels in both diaphragm and hindlimb muscles, while the alpha(2) and beta(2) isoforms increase postnatally and show greater muscle variation. beta(1) is the sole expressed beta-subunit in the diaphragm throughout development, and in the hindlimb muscles at birth. The Na,K-ATPase alpha(2) subunit is expressed during development when the t-tubules form. These results suggest that the alpha(2) isoform may serve, in part, a physiological role in the muscle t-tubules.