Hydrogen peroxide is now reported to be a normal aqueous humor component present, in the low microM concentration range, in the animal species which have been studied. This finding was established with the exclusive use of the dichlorophenol-indophenol method of analysis. In this procedure, aqueous humor is added to a blue, oxidized dichlorophenol-indophenol solution. The 605 nm absorbance of this solution immediately decreases in response to the reducing action of ascorbate present in the sample. The extent of reoxidation of the solution upon the addition of peroxidase, as measured by the increase in its 605 nm absorbance, can be quantitatively related to the concentration of H2O2 in the sample. A close examination of this method revealed that reduced dichlorophenol-indophenol spontaneously reoxidizes at a rate of 0.03 nmol min-1 microM-1, with generation of H2O2. H2O2 generation was unequivocally established by analysis of the temporal dependency of the absorbance increase produced by peroxidase in the absence of added H2O2 and by the sensitivity of this phenomenon to catalase. This spontaneous production of H2O2, on the other hand, cannot be attributed to ascorbate auto-oxidation because added ascorbate quantitatively reacts with dichlorophenol-indophenol, provided that an excess of the latter is maintained. This method then has an enormous potential to overestimate H2O2 in any sample. On the other hand, the response of the assay system to a given level of H2O2 depends on the level of reduction previously produced by ascorbate. This results in an artifactual positive correlation between ascorbate and H2O2 levels in samples containing variable amounts of ascorbate. In spite of these serious limitations the method can still be useful to measure H2O2 if appropriate precautions are taken. When using it for the analysis of rabbit aqueous humor H2O2 without correcting for the H2O2 generated during the assay and ignoring differences in the level of ascorbate in the samples, we obtained an average value of 25.3 microM H2O2, which coincides with that reported in the literature for the rabbit, but is obviously incorrect. When analysing aqueous humor there was the additional variable of the aqueous humor itself inhibiting the rate of dichlorophenol-indophenol auto-oxidation and so the final, corrected figure for H2O2 concentration in the aqueous humor became uncertain, since the auto-oxidation of the substrate could not be properly subtracted.(ABSTRACT TRUNCATED AT 400 WORDS)