We have characterized H(+)-translocating adenosine triphosphatase (ATPase) in membrane vesicles of Vibrio parahaemolyticus. The ATPase required high concentrations (about 0.5 M) of Na2SO4 (or other salts) for its maximum activity. Magnesium ion stimulated the ATPase activity, but Ca2+ did not. The activity of ATPase was inhibited by tetrachlorosalicylanilide, an H+ conductor, but not by another H+ conductor, carbonylcyanide-m-chlorophenylhydrazone. The activity was strongly inhibited by dicyclohexylcarbodiimide or Zn2+, and partially inhibited by azide, but not at all by vanadate.