Saposin (Sap) D is an endolysosomal protein that, together with three other similar proteins, Sap A, Sap B and Sap C, is involved in the degradation of sphingolipids and, possibly, in the solubilization and transport of gangliosides. We found that Sap D is able to destabilize and disrupt membranes containing each of the three anionic phospholipids most abundant in the acidic endolysosomal compartment, namely lysobisphosphatidic acid (LBPA), phosphatidylinositol (PI) and phosphatidylserine (PS). The breakdown of the membranes, which occurs when the Sap D concentration on the lipid surface reaches a critical value, is a slow process that gives rise to small particles. The Sap D-particle complexes formed in an acidic milieu can be dissociated by an increase in pH, suggesting a dynamic association of Sap D with membranes. The presence of anionic phospholipids is required also for the Sap D-induced perturbation and solubilization of membranes containing a neutral sphingolipid such as ceramide or a ganglioside such as G(M1). At appropriate Sap D concentrations Cer and G(M1) are solubilized as constituents of small phospholipid particles. Our findings imply that most functions of Sap D are dependent on its interaction with anionic phospholipids, which mediate the Sap D effect on other components of the membrane such as sphingolipids. On consideration of the properties of Sap D we propose that Sap D might have a role in the definition of the structure and function of membranes, such as the intra-endolysosomal membranes, that are rich in anionic phospholipids.