Lysosomal phospholipases play a critical role for degradation of cellular membranes after their lysosomal segregation. We investigated the regulation of lysosomal phospholipase A1 by cholesterol, phosphatidylethanolamine, and negatively-charged lipids in correlation with changes of biophysical properties of the membranes induced by these lipids. Lysosomal phospholipase A1 activity was determined towards phosphatidylcholine included in liposomes of variable composition using a whole-soluble lysosomal fraction of rat liver as enzymatic source. Phospholipase A1 activity was then related to membrane fluidity, lipid phase organization and membrane potential as determined by fluorescence depolarization of DPH, 31P NMR and capillary electrophoresis. Phospholipase A1 activity was markedly enhanced when the amount of negatively-charged lipids included in the vesicles was increased from 10 to around 30% of total phospholipids and the intensity of this effect depended on the nature of the acidic lipids used (ganglioside GM1<phosphatidylinositol approximately phosphatidylserine approximately phosphatidylglycerol approximately phosphatidylpropanol<phosphatidic acid). For liposomes containing phosphatidylinositol, this increase of activity was not modified by the presence of phosphatidylethanolamine and enhanced by cholesterol only when the phosphatidylinositol content was lower than 18%. Our results, therefore show that both the surface-negative charge and the nature of the acidic lipid included in bilayers modulate the activity of phospholipase A1 towards phosphatidylcholine, while the change in lipid hydration or in fluidity of membrane are less critical. These observations may have physiological implications with respect to the rate of degradation of cellular membranes after their lysosomal segregation.