This study was designed to elucidate the mechanisms underlying the formation of reactive oxygen species (ROS) by aminoglycoside antibiotics which may be causally related to the toxic side effects of these drugs to the kidney and the inner ear. ROS formation by aminoglycosides in vitro requires iron and the presence of polyunsaturated lipids as electron donors. Electron spray ionization mass spectrometry (ESI-MS) confirmed earlier observations that gentamicin strongly binds to l-α-phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂), a membrane lipid rich in arachidonic acid. Studies using lipid-coated membranes (PIP strips) further indicated that iron ions and gentamicin can simultaneously bind to phosphoinositides with at least one phosphate group on the inositol ring, suggesting the existence of ternary complexes among gentamicin, iron, and phospholipids. Peroxidation of PtdIns(4,5)P₂ by ferrous ions significantly increased in the presence of gentamicin, and EI-MS measurements indicated that oxidative damage to PtdIns(4,5)P₂ was accompanied by the release of arachidonic acid. Arachidonic acid also forms a ternary complex with Fe^2+/3+−gentamicin, confirmed by ESI-MS, that reacts with lipid peroxides and molecular oxygen, leading to the propagation of arachidonic acid peroxidation.