Abstract Hydroperoxide lyases (HPL E.C. 4.1.2.) are part of the lipoxygenase pathway in plants and catalyze the conversion of fatty acid hydroperoxides into oxo acids and short chain aldehydes. These aldehydes have desirable properties for the food and agricultural industry. HPL activity can be modulated by salts and surfactants, but the mechanisms governing the modulation are not fully understood. Recombinant HPL activity was evaluated by use of factorial experimental design investigating the effects of KCl and Triton X-100 on HPL activity with 13-hydroperoxy-octadecadienoic acid (LA-OOH) and 13-hydroperoxy-octadienoyl sulfate (LS-OOH) as substrates. To investigate solubility issues of the two different substrates, an aqueous and a two-phase micro-aqueous reaction medium was used. The highest HPL activity (8.7 μmol min −1 mg −1 ) was achieved under aqueous conditions with high salt (1.5 M) and low surfactant (0%, v/v) concentrations and LA-OOH as a substrate. Maximal activity (2.4 μmol min −1 mg −1 ) under micro-aqueous conditions was achieved with high salt (1.5 M) and high surfactant (0.01%, v/v) concentrations and LS-OOH as a substrate. A significant interaction between salt and surfactant as well as salt and substrate could be identified and a hypothesis for the interaction phenomena is presented.