Retinal ganglion cells (RGCs) undergo apoptosis after axonal injury. Elucidation of the sequence of intracellular events proximal to caspase activation may allow development of effective neuroprotective strategies. In this study, we explored the role that reactive oxygen species may have in signaling RGC apoptosis after axonal injury. Using the fluorescent probe dihydroethidium, we were able to measure intracellular superoxide anion production. We found that axotomized RGCs exposed to oxidative stress exhibited a secondary superoxide burst. The broad-spectrum caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethyl ketone did not block the burst, suggesting it is proximal to caspase activation, but it was inhibited by cycloheximide, consistent with a requirement for protein synthesis. These results are consistent with RGC axotomy inducing synthesis of one or more proteins that mediate oxidative amplification. This could be an early event in signaling of RGC apoptosis after axonal injury.