The capacity of H2O2, the most stable of the reactive oxygen species (ROI), to diffuse freely across biological membranes and to signal gene expression suggests that H202 could function as a short-lived second messenger diffusing from cell to cell. We tested this hypothesis in tobacco plants treated with a glycoprotein elicitor. Applied at 50 nM, it induces H2O2 accumulation and the hypersensitive response restricted to the infiltrated zone 1 tissue. Stimulation of a set of defense responses also occurs in the surrounding zone 2 tissue without diffusion of the elicitor. ROI levels in zone 1 were modulated using N-acetyl-L-cysteine (NAC) as a ROI scavenger and Rose Bengal (RB) as a ROI generator. We found that ROI appeared to act as signalling intermediates in pathways leading to salicylic acid accumulation, to PR1, PR5 and 3-hydroxy-3-methylglutarylCoA reductase expression in glycoprotein-treated zone 1 tissues. Compared to the treatment with the elicitor alone, coinfiltration of the glycoprotein and NAC increased the surface of zone 2 showing PR1 and O-methyltransferase expression. Application of RB had the opposite effect. The data suggest that, in our system, ROI did not act as a cellto-cell diffusible signal to activate PR protein and O-methyltransferase expression in zone 2.