Guinea pig liver microsomal membranes were cholesterol-enriched by feeding guinea pigs a high-cholesterol diet. Cholesterol enrichment as well as partial lipid removal of normal native microsomes by acetone-butanol extraction resulted in 40-50% loss in activity of the glucose-6-phosphate phosphohydrolase (G-6-Pase) (EC 3.1.3.9) enzyme system. The activity was restored by supplementation of microsomal total phospholipid (PL) and its phosphatidylcholine (PC) species but not with microsomal neutral lipids, cholesterol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin or diphosphatidylglycerol (cardiolipin). The activity was decreased by sodium deoxycholate but enhanced by dimethylsulfoxide. Egg-yolk PC and asolectin influenced the activity of the enzyme to the same extent as microsomal PC did. Lipid depletion and cholesterol produced an increase in Km while the Vmax was lowered. The non-linearity in the Arrhenius plot of the native microsomes was lost on lipid removal and cholesterol enrichment. The energy of activation (Ea) calculated from the continuous line was found to be lowered to the level that was observed above the break points in intact microsomes. Addition of microsomal PC to the assay system decreased the Km of the enzymatic reaction in native membranes, in partially lipid-depleted and cholesterol-enriched membranes, but did not alter the Vmax values and only marginally influenced the non-linear relationship of the Arrhenius expression of temperature dependence. The ability of immature rat liver phospholipid exchange protein to introduce alien PL into microsomal membrane was used to study the lipid dependence of G-6-Pase. Protein-catalyzed and detergent (cholate)-mediated membrane PL exchange for egg-yolk PC from the PC/cholesterol unilamellar liposomes resulted in substantial loss of enzyme activity. The discrepancies in the influence of PC on G-6-Pase were interpreted by assuming that the enzyme was a two-component system, a surface-located substrate transporter unit and a membrane integral catalytic phosphohydrolase unit. The lipid microenvironment and PL requirement in particular, could be different for the two components, although they represented a single functional unit at the time of enzymatic reaction.