We have examined the authenticity of two methods for determination of H 2 O 2 in leaf tissue. We show that the high concentrations of ascorbic acid present in leaf extracts interfere with both techniques. In the chromogenic peroxidase-coupled assay, H 2 O 2 is determined by oxidation of 3-methyl-2-benzothiazoline hydrazone (MBTH) and 3-(dimethylamino) benzoic acid (DMAB). The method yields two phases of absorbance increase as these substrates are oxidized. We show (a) that only the first phase is dependent on extracted H 2 O 2 ; (b) that the slow phase is due to phenolic-dependent generation of H 2 O 2 during the assay; and (c) that ascorbate inhibits both phases. These effects could explain both the high values and the variable results found in the literature. The chemiluminescence method involves H 2 O 2 enhancement of ferricyanide-induced chemiluminescence of luminol (3-amino-phthal-hydrazide). Chemiluminescence of luminol is strongly inhibited by added ascorbate, suggesting that failure to remove ascorbate from extracts will cause this method to underestimate H 2 O 2 . Using the fast phase of the peroxidase-coupled assay to estimate H 2 O 2 in extracts from which ascorbate and phenolic compounds had been removed, we obtained leaf contents of H 2 O 2 within the range of 40-120 nmol g - 1 FW.