Specific functions served by the various Na⁺-K⁺-ATPase -isoforms are likely to originate in regions of structural divergence within their primary structures. The isoforms are nearly identical, with the exception of the NH₂ terminus and a 10-residue region near the center of each molecule (isoform-specific region; ISR). Although the NH₂ terminus has been clearly identified as a source of isoform functional diversity, other regions seem to be involved. We investigated whether the central ISR could also contribute to isoform variability. We constructed chimeric molecules in which the central ISRs of rat ₁- and ₂-isoforms were exchanged. After stable transfection into opossum kidney cells, the chimeras were characterized for two properties known to differ dramatically among the isoforms: their K⁺ deocclusion pattern and their response to PKC activation. Comparisons with rat full-length ₁- and ₂-isoforms expressed under the same conditions suggest an involvement of the central ISR in the response to PKC but not in K⁺ deocclusion.