Specific functions served by the various Na⁺-K⁺-ATPase isoforms are likely to originate within regions of structural divergence within their primary structures. The isoforms are nearly identical, with the exception of the amino-terminus and a ten-residue region near the center of each molecule. Although the amino-terminus has clearly been identified as a source of isoform functional diversity, other regions seem to be involved. We investigated whether the ten-residue region near the center of the molecule (Isoform Specific Region: ISR) could also contribute to isoform variability. We constructed chimeric molecules in which the central ISR of rat 1 and 2 isoforms have been exchanged. After stable transfection into opossum kidney (OK) cells, the chimeras were characterized for two properties known to differ dramatically among the isoforms, their K⁺ deocclusion pattern and their response to protein kinase C (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.