Aminoglycoside antibiotics accumulate in lysosomes of kidney and cultured cells and cause an impairment of phospholipid catabolism which is considered to be an early and significant step in the development of their toxicity. Using liposomes, wer previously demonstrated that the activity of lysosomal phospholipases A1 and A2 towards phosphatidylcholine was markedly enhanced by the inclusion of phosphatidylinositol in the bilayer, and that gentamicin impaired this activity by binding to phosphatidylinositol. Since gentamicin-induced inhibition was inversely related to the amount of phosphatidylinositol included in the liposomes, we proposed that gentamicin impairs activity of phospholipases by decreasing the quantity of available negative charges carried by the bilayer surface (Mingeot-Leclercq et al., Biochem Pharmacol 37: 591-599, 1988). We now extend these observations to phosphatidylserine and phosphatidic acid, and compare the inhibition caused by gentamicin, amikacin and streptomycin towards lysosomal phospholipases on the hydrolysis of phosphatidylcholine in the presence of each of these acidic phospholipids. Inclusion of phosphatidic acid in liposomes, and, to a lesser extent, phosphatidylserine, caused a larger increase in phospholipases activity than phosphatidylinositol. In parallel, the three aminoglycosides tested were found less inhibitory towards phospholipases activity measured on phosphatidic acid-or phosphatidylserine-containing liposomes than was previously observed with phosphatidylinositol, even though equilibrium dialysis experiments failed to demonstrate significant difference in binding parameters of the drug towards each of these liposomes populations. Yet, as for phosphatidylinositol-containing liposomes, the inhibition was inversely related to the amount of phosphatidic acid or phosphatidylserine included in the bilayer and the inhibitory potency of the three drugs was consistently gentamicin greater than amikacin greater than streptomycin with the three types of negatively-charged liposomes used. We conclude that impairment of lysosomal phospholipases activity towards phosphatidylcholine included in negatively-charged membranes by aminoglycoside antibiotics is dependent upon drug binding to the bilayer, but that it is modulated by the nature of the acidic phospholipid that binds the drug as well as by that of the drug itself. A companion paper (Mingeot-Leclercq et al., Biochem Pharmacol 40: 499-506, 1990) will examine by computer-aided conformational analysis the parameters (drug-phospholipid energy of interaction, position of the drug in a monolayer and its accessibility to the aqueous phase) which may be important for these effects.