The present study was designed to test the hypothesis that the release of neutrophil chemotactic factors (NCF) from isolated corneas following hydrogen peroxide stimulation requires specific intracellular synthesis. For these studies, the epithelial surfaces of isolated rabbit corneas were preincubated with various inhibitors of protein synthesis (cycloheximide, 10μg/ml, and puromycin, 50μg/ml) and transcription (actinomycin D,μg/ml) for 60 min prior to exposure of the corneas to glucose (G, 1 mg/ml) and glucose oxidase (GO, 20 U/ml) for 6 h at 37°C. All three inhibitors decreased the levels of NCF recovered in the extracorneal fluids by 80–98%, suggesting that peroxide acts to upregulate NCF production at both the transcription and translation levels. When corneas were incubated with G/GO for 6 h at 12°C or 4°C instead of 37°C, a reduction in the levels of NCF recovered in the supernatants was noted at 12°C (46–91% inhibition) and at 4°C (67–96% inhibition), suggesting that the synthesis of NCF at cold temperature was only reduced but not totally inhibited. To demonstrate that the observed reduction in chemotactic activity recovered from corneas incubated at 12°C or 4°C is not due to a temperature-dependent inhibition of NCF biosynthesis, but rather to a disruption of intracellular vesicular transport, temperature shift experiments were performed. Corneas were incubated with G/GO overnight at 12°C or 4°C prior to shifting to 37°C for an additional 6 h. Two peaks of activity were recovered, one immediately (1 min) after shifting the corneas to 37°C, and the second after 6 h of incubation at 37°C, consistent with our previous findings. Corneas continuing incubation at 12°C or 4°C, on the other hand, released no chemotactic factors, suggesting that the exocytosis of NCF synthesized overnight was disrupted. These studies support the conclusion that NCF released from hydrogen peroxide-injured corneas require biosynthesis of specific intracellular proteins.