In a previous study we indicated that the α subunit of the Na,K-ATPase has 4 transmembrane segments in the COOH terminal domain between residues Lys769 and Val939, and that both the NH2-terminus and the COOH-terminus are in the cytosol. However, there was insufficient information to determine whether there are more transmembrane segments between residues Val939 and the COOH-terminal Tyr1018. To investigate this question, we inserted the influenza virus hemagglutinin (HA)-epitope between Leu973 and Arg974 of the α1 chain, expressed the construct in COS-7 and HeLa cells and determined the membrane arrangement by indirect immunofluorescence. The results indicate that Leu973 is not on the extracellular surface of the plasma membrane. Thus, the α1 subunit is likely to possess only four complete transmembrane segments in the COOH terminal domain between residues Lys769 and Tyr1018.