Models of P-type ATPase predict that membrane-embedded fragments represent about 20% of the protein and adopt an all-α-helical structure. While this prediction was confirmed for the Ca2+ -ATPase [Corbalan-Garcia, S., Teruel, J., Villalain, J. and Gomez-Fernandez, J. (1994) Biochemistry 33, 8247-8254], it is at odds with recent experimental evidence gathered on the Neurospora crassa plasma membrane H+ -ATPase [Vigneron, L., Ruysschaert, J.-M. and Goormaghtigh, E. (1995) J. Biol. Chem. 270, 17685-17696] and on the gastric H+,K+ -ATPase [Raussens, V., Ruysschaert, J.-M. and Goormaghtigh, E. (1997) J. Biol. Chem. 276, 262-270]. Extensive proteinase K proteolysis of open gastric tubulovesicles was performed here to generate the membrane-protected fragments of the H+,K+ -ATPase. Secondary structure of the intact and of the membrane-protected segments was compared for oriented membrane films by attenuated total-reflection Fourier-transform infrared spectroscopy and by circular dichroism and for vesicles suspension by circular dichroism and Raman spectroscopy. All the spectroscopic data indicate that the protease-resistant membrane-bound residue of the H+,K+ -ATPase contains significant amount of β-sheet structure, both on films and in membrane suspensions. Polarized attenuated total-reflection infrared spectroscopy indicates that only the α-helical content of protease-resistant membrane-bound residue of the H+,K+ -ATPase is oriented (parallel) with respect to the membrane normal. Raman spectroscopy reveals that Phe residues are preferentially removed by protease activity. Evaluation of the amount of removed Phe and Tyr residues places constraints on the model of membrane insertion of the H+,K+ -ATPase.