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Several mutations of residues Glu795and Glu820present in M5 and M6 of the catalytic subunit of gastric H+,K+-ATPase have resulted in a K+-independent, SCH 28080-sensitive ATPase activity, caused by a high spontaneous dephosphorylation rate. The mutants with this property also have a preference for theE1conformation. This paper investigates the question of whether these two phenomena are coupled. This possibility was studied by combining mutations in residue Glu343, present in M4, with those in residues 795 and 820. When in combined mutants Glu and/or Gln residues were present at positions 343, 795, and 820, the residue at position 820 dominated the behavior: a Glu giving K+-activated ATPase activity and anE2preference and a Gln giving K+-independent ATPase activity and anE1preference. With an Asp at position 343, the enzyme could be phosphorylated, but the dephosphorylation was blocked, independent of the presence of either a Glu or a Gln at positions 795 and 820. However, in these mutants, the direction of theE2↔ E1equilibrium was still dominated by the 820 residue: a Glu givingE2and a Gln giving E1. This indicates that the preference for the E1conformation of the E820Q mutation is independent of an active dephosphorylation process.