We studied the topogenic properties of five hydrophobic segments (H5-H9) in the COOH-terminal third of Na,K-ATPase ␣ subunit using in vitro insertion of fusion proteins into endoplasmic reticulum membranes. These fusion proteins consisted of several different lengths of truncated ␣ subunit starting at Met 729 and a reporter protein, chloramphenicol acetyltransferase, that was linked in frame after each hydrophobic segment. We found that membrane insertion of the newly synthesized COOH-terminal third was initiated by H5 and terminated by H9, indicating that here only H5 and H9 have topogenic function. The other three, H6 -H8, did not have topogenic function in the native context and were translocated into the endoplasmic reticulum lumen. These results were in striking contrast to the previous models in which four or six hydrophobic segments were proposed to cross the membrane. Furthermore, the findings suggest a novel mechanism for achieving the final membrane topology of the COOH-terminal third of the ␣ subunit.Na,K-ATPase consists of two non-covalently linked subunits: a larger non-glycosylated ␣ subunit (about 100 kDa) and a glycosylated  subunit (approximately 55 kDa) (Brotherus et al., 1983), both of which are transmembrane proteins. The complete amino acid sequence of the ␣ subunit from different sources has been cloned (see review by Mercer (1993)). Hydropathy analysis of the sequence has shown nine hydrophobic segments (H1-H9). Four segments (H1-H4) are in the NH 2 -terminal third and the rest (H5-H9) are in the COOH-terminal third of the subunit. However, H5 is often proposed to cross the membrane twice as H5 and H6, so that the remaining segments are often named H7-H10.The four hydrophobic segments in the NH 2 -terminal third have been demonstrated to be transmembrane spans by immunochemical studies (Kano et al