Aminoglycosides exert their cytotoxic effect at three different locations: at the cell surface, in the cytosol, or in the nucleus. At the cell surface, aminoglycoside binding can cause temporary hearing loss, motor paralysis at the neuromuscular junction, ion wasting in kidneys, or analgesia in mechano- and nocioreceptors (touch and pain sensory cells). More severe and permanent cytotoxic events occur following aminoglycoside entry into the cell, via one or more transport mechanisms to cross the plasma membrane. Endocytosis is the most widely studied aminoglycoside uptake mechanism, but it remains uncertain whether this is receptor-mediated, non-specific fluid-phase mediated, or both; and if endocytotic uptake can lead directly to cytotoxicity. Endocytosis is a rate-limiting, temperature-dependent mechanism that does not account for the rapidity or temperature-independence of observed cytotoxic sequelae, including mitochondrial permeabilization, and toxic generation of reactive oxygen species. Aminoglycosides can also enter directly into the cytosol through ion channels in auditory and vestibular hair cells, kidney cells and other sensory neurons. This rapid, cell-selective mechanism of uptake can lead to rapid, direct interaction or binding with (1) phosphatidylinosito l4,5-bisphosphate (PIP[subscript 2]) that could generate endogenous agonists of aminoglycoside permissive channels (2) mitochondria leading to generation of reactive oxygen species; (3) ribosomes and mistranslation during protein synthesis; and (4) RNA-composed nucleoli within the nucleus of cells. Potential mechanisms by which aminoglycosides are transported across the blood-labyrinth barrier into the endocochlear fluids, clearance from cells, and synergism with loop diuretics are also discussed.