Aminoglycoside antibiotics are very effective against severe Gram-negative infections, but their clinical use is associated with nephrotoxic side-effects. The cascade of events leading to acute renal failure involves an impairment of lysosomal phospholipase activity, which is thought to result from the direct interaction of the drugs with the head group of negative phospholipids. Fourier transform infrared spectroscopy was used to study the effects of three aminoglycosides from the kanamycin family (amikacin and kanamycins A and B) on dimyristoylphosphatidylglycerol (DMPG) bilayers at lysosomal pH. The results obtained were consistent with a tightening of the lipidic network caused by the neutralization of the negative head groups of DMPG by the positive charges of the aminoglycosides. These antibiotics induced an increase of the transition temperature of DMPG, a decrease of both the frequency and relative intensity of the hydrogen-bonded carbonyl component, and a decrease of the phosphate antisymmetric band frequency. Kanamycin B, which is known to be the most nephrotoxic drug of the three, exhibited the greatest effects on the transition temperature and on the carbonyl stretching band. A comparison of the nature and extent of the spectral changes led us to conclude that amikacin lies flat on the bilayer surface, whereas kanamycin B is located between the lipidic head groups and quite close to some of the carbonyl groups. Finally, a possible correlation between the importance of bilayers perturbation and the respective inhibitory potency against phospholipases was examined.