This study uses genetically altered mice to examine the contribution of the Na,K-ATPase 2 catalytic subunit to the resting potential, excitability, and contractility of the perinatal diaphragm. 2 protein is reduced by 38% in the 2 heterozygous and absent in the 2 knockout, and 1 is upregulated 1.9 fold in the knockout. Resting potentials are depolarized by 1.8 - 4.0 mV in the heterozygous and knockout. Action potential threshold, overshoot, and duration are normal. Spontaneous firing, a developmental function, is impaired in the knockout diaphragm but this does not compromise its ability to fire evoked action potential trains, the dominant mode of activation near birth. Maximum tetanic force, rate of activation, force-frequency and force-voltage relationships, and onset and magnitude of fatigue are not changed. The major phenotypic consequence of reduced 2 content is that relaxation from contraction is 1.7 fold faster. This reveals a distinct cellular role of the 2 isoform which cannot be restored simply by increasing 1 content. Na/Ca exchanger expression decreases in parallel with 2, suggesting that Ca²⁺ extrusion is affected by the altered 2 genotype. There are no major compensatory changes in expression of sarcoplasmic reticulum Ca-ATPase, phospholamban, or the plasma membrane Ca-ATPase. These results demonstrate that the Na,K-ATPase 1 isoform alone is able to maintain equilibrium K⁺ and Na⁺ gradients and to substitute for 2 in most cellular functions related to excitability and force. They further demonstrate that the 2 isoform contributes significantly less at rest than expected from its proportional content, but can modulate contractility during muscle contraction.