Generation of hydrogen peroxide (H₂O₂) by chemicals in mainstream cigarette smoke is of prime interest for the estimation of the pro-oxidant activity of the smoke. Since most of phenolic compounds (potential H₂O₂ precursors) reside in the particulate phase of the cigarette smoke, the total particulate matter (TPM) generates a larger amount of hydrogen peroxide compared to the gas phase of the smoke. To date there are no data in the literature which reveal the influence of the gas phase of cigarette smoke on the ability of the particulate phase to generate H₂O₂. The key part of the H₂O₂ generation mechanism involves the formation of superoxide species from appropriate semiquinone reactants in particulates. These superoxide intermediates undergo spontaneous dismutation to afford a limited amount of hydrogen peroxide. Conversely, catalysis by superoxide dismutase (SOD) results in a significantly enhanced H2O2 yield. It is possible that the smoke gas phase reduces the SOD activity, thus inhibiting H₂O₂ formation. To investigate this mechanism, we used commercially sourced cigarettes (BAT Russia; with TPM delivery at 11.0 ± 0.5 mg), which were smoked by a A14 single-port machine (Borgwaldt KC GmbH), while the gas and the particulate phases of the smoke were separated by Cambridge filter pads. The developed chemiluminescence assay for H₂O₂ in experimental samples utilised luminol as the chemiluminophore and horseradish peroxidase (HRP) (Dia-M, 400 units/mg) as the catalyst. The sensitivity of such an assay enabled measuring as little as 0.5 ng/ml of H2O2 in the sample. We found that the gaseous phase smoke significantly affects H2O2 generation by the TPM (thus, gas-phase condensate of the smoke collected from eight cigarettes caused more than a two-fold decrease of the TPM-derived H2O2 level).