The aim of this study was to determine if changes in protein content and/or gene expression of Na⁺-K⁺ ATPase subunits underlie its decreased enzyme activity during ischemia and reperfusion. We measured protein and mRNA subunit levels in isolated rat hearts subjected to 30 min of ischemia and 30 min of reperfusion (I-R). The effect of ischemic preconditioning (IP), induced by three cycles of ischemia and reperfusion (10 min each), was also assessed on the molecular changes in Na⁺-K⁺ ATPase subunit composition due to I-R. I-R reduced the protein levels of the ₂, ₃, ₁, and ₂ isoforms by 71, 85, 27 and 65% respectively, whereas the ₁ isoform was decreased by <15%. A similar reduction in mRNA levels also occurred for the isoforms of Na⁺-K⁺ ATPase. IP attenuated the reduction in protein levels of Na⁺-K⁺ ATPase ₂, ₃, and ₂ isoforms induced by I-R, without affecting the ₁ and ₁ isoform. Furthermore, IP prevented the reduction in mRNA levels of Na⁺-K⁺ ATPase ₂, ₃, and ₁ isoforms following I-R. Similar alterations in protein contents and mRNA levels for the Na⁺-Ca²⁺ exchanger were seen due to I-R as well as IP. These findings indicate that remodeling of Na⁺-K⁺ ATPase may occur due to I-R injury and this may partly explain the reduction in enzyme activity in ischemic heart disease. Furthermore, IP may produce beneficial effects by attenuating the remodeling of Na⁺-K⁺ ATPase and changes in Na⁺-Ca²⁺ exchanger in hearts after I-R.