The Na,K-ATPase α2 isoform in skeletal muscle is rapidly stimulated during muscle use and plays a critical role in fatigue resistance. The acute mechanisms which stimulate α2 activity are not completely known. This study examines whether phosphorylation of phospholemman (PLM/FXYD1), a regulatory subunit of Na,K-ATPase, plays a role in the acute stimulation of α2 in working muscles. Mice lacking PLM (PLM KO) have a normal content of α2 subunit and show normal exercise capacity, in contrast to the greatly reduced exercise capacity of mice which lack α2 in the skeletal muscles. In PLM KO mice, neither nerve-evoked contractions in vivo or fatiguing contractions of isolated muscles induces a change in total PLM or PLM phosphorylated at Ser63 or Ser68. Isolated muscles of PLM KO mice maintain contraction and resist fatigue as well as WT. Rb⁺ transport by the α2 Na,K-ATPase is stimulated to the same extent in contracting WT and contracting PLM KO muscles. Phosphorylation of sarcolemmal membranes prepared from WT but not PLM KO skeletal muscles stimulates the activity of both α1 and α2 in a PLM-dependent manner. The stimulation occurs by an increase in Na⁺ affinity without significant change in V_max, and is more effective for α1 than α2. These results demonstrate that phosphorylation of PLM is capable of stimulating the activity of both isozymes in skeletal muscle; however, contractile activity alone is not sufficient to induce PLM phosphorylation. Importantly, acute stimulation of α2, sufficient to support exercise and oppose fatigue, does not require PLM or its phosphorylation.