Mg￣(2+)-ATPase had been partly purified from Acholeplasm Laidlawii AIH089membranes by extraction with 1% DOC followed by Separose-6B and DEAE-Cellulose DE52 chromatography. The Mg￣(2+)-ATPase was then successfully reconstituted with a number of different phospholipids and glycolipids using a cholate solubilation and dialysis procedure.The ability of these lipids to support the function of this enzyme was evaluated by their ability to increase the specific activity of the enzyme. The incorporation of the ATPase into liposomes composed of the endogenous membrane glycolipids (MGDG and DGDG), zwitterionic phospholipid (PC) or nonbilayer phospholipid (PE), resulted in full recovery of the enzyme activity. In contrast,anionic phospholipid alone, such as PG, DPG, would cause inhibition to this ATPase. Our results indicated that Mg￣(2+)-ATPase from A. laidlawii membranes is unlike the other ATPase, including Ca￣(2+)-ATPase from sarcoplasmic reticulum; (Na￣+K￣+)ATPase from rabbit brain and H￣+-ATPase from mitochondria membranes, which are dependent on anionic phospholipids rather than zwitterionic phospholipids from their activity.