Cellular stress results in the activation of members of the Mitogen Activated Protein Kinase (MAPK) family including c-Jun N-terminal kinases (JNK), p38 kinases and Extracellularly Regulated Kinases 1 and 2 (ERK1/2). JNK signaling is known to play a role during sensory hair cell death in the inner ear (e.g. Wang et al, 2003). Here, we investigated ERK1/2 activation in the inner ear using two different damage paradigms. Cochlear cultures were prepared from postnatal day 1 rat cochleae and used after 1 day in vitro. The sensory hair cell epithelium was lesioned using a N2-laser which allowed precise timing of the damage onset. ERK1/2 activation was assessed using an antibody directed to the dually-phosphorylated form of ERK1/2. Laser lesioning induced the transient and specific activation of ERK1/2 in support cells but not in hair cells. The signal spread non-uniformly from the lesion site with maximum spread occurring 10 minutes after damage. Aminoglycoside antibiotics are known to induce hair cell death both in vitro and in vivo (Wang et al, 2003). We treated cochlear cultures with neomycin (1mM) for 8 or 24 hrs to test whether ERK1/2 was also activated when hair cells were specifically targeted with aminoglycosides. Cultures were triple labeled with (i) a DNA stain; (ii) the activated ERK1/2 antibody and (iii) fluorescent phalloidin to label actin rich stereocilia at the apical surface of hair cells. Hair cell death was quantified by counting pyknotic nuclei. Treatment with neomycin for 8 hrs caused a significant increase in pyknotic inner hair cell nuclei compared to untreated controls. At this time point, ERK1/2 activation was observed in clusters of support cells surrounding dying hair cells. By 24 hrs, pyknotic outer hair cell nuclei were observed and again ERK1/2 were activated in the support cells. U0126, an inhibitor of MEK1/2, the MAPK kinase was used to test whether the activation of ERK1/2 plays a role in neomycin-induced hair cell death. At 8 hrs, neomycin induced inner hair cell death was significantly decreased in the presence of 10 µM U0126. However after 24 hrs, this protective effect was overcome and there was no significant difference between U0126-treated and untreated cultures. In outer hair cells a reduction in cell death was observed at both 8 and 24 hrs in the presence of U0126; however the effect was not statistically significant. These data suggest that ERK1/2 activation in support cells is a common signalling mechanism during hair cell damage and this activation can contribute to the subsequent death of hair cells.