At two intramembrane carboxyl-containing amino acids of the sheep al isoform of Na,K-ATPase (Asp 804 and Asp 808) both charge-conserving (Asp to Glu) and charge-deleting (Asp to Asn, Leu and Ala) replacements were made and the altered enzymes studied. Nucleotide changes encoding the amino acid substitutions were placed in a cDNA encoding a ouabainresistant enzyme (sheep α1 RD) and the encoded enzymes were expressed in ouabain-sensitive HeLa cells. Transfections with cDNAs carrying all Asp 804 substitutions, along with those carrying As 808Ala, Asp 808 Asn, and Asps 808Leu replacements failed to confer ouabain resistance to the cells, indicating critical roles for Asp 804 and Asps 808. Only the expression of the Asp 808Glu enzyme produced ouabain-resistant HeLa cells, demonstrating that the altered protein was functional. When the inactive proteins Asp 804Ala and Asp 080Ala were expressed using an alternative selection system (the protein carrying the amino acid substitution was the ouabain-sensitive wild-type sheep αl Na,K-ATPase, which was expressed in ouabain-resistant 3T3 cells), intact cells were able to bind extracellular ouabain with high affinity ( K d =1–30 nM), indicating that the inactive proteins were synthesized and folded properly in the plasma membrane. The results demonstrate that carboxyl side chains at positions 804 and 808 are critical for enzyme catalytic function.