Aminoglycoside antibiotics cause aggregation but not fusion of negatively-charged liposomes at an extent proportional to their capacity to interact with acidic phospholipids (Van Bambeke et al., 1995, Eur. J. Pharmacol., 289, 321-333). To understand why aggregation is not followed by fusion, we have examined here the influence of two aminoglycosides with markedly different toxic potential (gentamicin > isepamicin) on lipid phase transition in negatively-charged liposomes using 31P-NMR spectroscopy, in comparison with spermine (an aggregating agent) and bis(beta-diethylaminoethylether)hexestrol or DEH (a fusogenic cationic amphiphile). Gentamicin, spermine, and, to a lesser extent, isepamicin inhibit the appearance of the isotropic signal seen upon warming of control liposomes and denoting the presence of mobile structures. This non-bilayer signal appeared most prominently when liposomes were incubated with DEH, a strong fusogenic agent. We conclude that aminoglycosides, like spermine, have the potential to prevent membrane fusion, by inhibiting the development of a critical change in membrane organization, which is associated with fusion. We suggest that this capacity could be a determinant in aminoglycoside toxicity.