Aminoglycoside treatment induces caspase-dependent apoptotic death in inner ear sensory hair cells. The timing of apoptotic signaling in sensory hair cells following systemic aminoglycoside treatment has not been characterized in vivo. We administered a single subcutaneous injection of the aminoglycoside gentamicin (300 mg/kg) to 12–16-day-old chicks and used immunocytochemical techniques to document the following responses in affected hair cells: T-cell restricted intracellular antigen-related protein (TIAR) translocation from the nucleus to the cytoplasm, cytochrome c release from the mitochondria, caspase-3 activation, nuclear condensation, and an orderly progression of hair cell ejection from the proximal end of the basilar papilla. Hair cells in the proximal tip exhibited TIAR translocation from the nucleus and aggregation into punctate granules in the cytoplasm 12 hours after injection and the response progressed distally. Cytochrome c release from the mitochondria into the cytoplasm and caspase-3 activation were observed in affected hair cells immediately prior to and during ejection. Hair cell ejection occurred between 30 and 54 hours after injection, beginning in the proximal tip and progressing distally. Nuclear condensation accompanied ejection while the loss of: 1) membrane integrity; 2) phalloidin labeling of F-actin; and 3) TO-PRO-1 labeling of nuclear contents occurred within 48 hours following ejection. Our results present a timeline of aminoglycoside-induced inner ear sensory hair cell apoptotic death that includes an 18-hour window between the initial apoptotic response and the later stages of programmed death signaling that accompany ejection and a gradual breakdown of hair cells following ejection. J. Comp. Neurol. 475:1–18, 2004. © 2004 Wiley-Liss, Inc.