The Na,K-ATPase is essential for the contractile function of skeletal muscle, which expresses the a1 and a2 subunit isoforms of Na,K-ATPase. The a2 isozyme is predominant in adult skeletal muscles and makes a greater contribution in working compared with noncontracting muscles. Hindlimb suspension (HS) is a widely used model of muscle disuse that leads to progressive atrophy of postural skeletal muscles. This study examines the consequences of acute (6–12 h) HS on the functioning of the Na,K-ATPase a1 and a2 isozymes in rat soleus (disused) and diaphragm (contracting) muscles. Acute disuse dynamically and isoform-specifically regulates the electrogenic activity, protein, and mRNA content of Na,K-ATPase a2 isozyme in rat soleus muscle. Earlier disuse-induced remodeling events also include phospholemman phosphorylation as well as its increased abundance and association with a2 Na,K-ATPase. The loss of a2 Na,K-ATPase activity results in reduced electrogenic pump transport and depolarized resting membrane potenti