The binding of aminoglycoside antibiotics to acidic phospholipids of membranes is an essential step in the development of both their renal and auditory toxicities, which could be associated with critical modifications of the membrane properties. This work examines the capacity of aminoglycosides to induce membrane aggregation and fusion. Three techniques were used in parallel: (i) measurement of the dequenching rate of a lipid-soluble fluorescent probe (octadecylrhodamine B) incorporated at self-quenched concentration in membranes; (ii) measurement of the increase in the energy transfer between two fluorescent derivatives of phospholipids; and (iii) electron microscopy of negatively-stained replicas. The results were compared with those obtained with spermine (an aggregating polycation) and melittin (a fusogenic peptide). The three approaches indicate that aminoglycosides induce liposomes aggregation, but not fusion. Aggregation is related to the capacity of each drug studied to bind phosphatidylinositol, as evaluated by its energy of interaction with this acidic phospholipid, and to its toxic potential. Membrane aggregation occurring in vivo could therefore contribute to, or be a determinant of this toxicity, which could rationally be screened for new derivatives by the methods applied here.