The Na,K-ATPase is a membrane-associated enzyme that establishes the internal Na+/K+ environment of most animal cells. The catalytic (alpha) subunit of the Na,K-ATPase contains multiple transmembrane segments, but the number and location of these domains has not been clearly established. We have used epitope addition to determine the transmembrane topology of the alpha subunit. An immunoreactive peptide was inserted into various regions of the cDNA encoding the rat alpha 1 subunit, and the constructs were expressed in transfected mammalian cells. The intra- or extracellular location of the epitope tags was determined by immunofluorescence analysis. Our results indicate that the amino and carboxyl termini of the alpha subunit are situated intracellularly, and the polypeptide is likely to possess eight membrane-spanning segments. The systematic application of epitope tagging may be useful for analyzing the topology of membrane proteins of unknown structure.