Phagocytes kill ingested microbes by exposure to high concentrations of toxic reactive species generated by NADPH-oxidases. This membrane-bound electron-transferring enzyme is tightly regulated by cellular signaling cascades. So far, molecular and biophysical studies of the NADPH-oxidase were performed over limited temperature ranges, which weaken our understanding of immune response or inflammatory events. In this work, we have inspected the influence of temperature and lipid membrane properties on the NADPH-oxidase activity using a system free of cell complexity.We have extended the experimental conditions of the accepted model for NADPH-oxidase activity, the so-called cell-free assay, to a large temperature range (10-40°C) using different membrane compositions (subcellular compartments or liposomes).A remarkable increase of superoxide production rate was observed with rising temperature. Synchrotron radiation circular dichroism data showed that this is not correlated with protein secondary structure changes. When lipid bilayers are in fluid phase, Arrhenius plots of the oxidase activity showed linear relationships with small activation energy (Ea), while when in solid phase, high Ea was found. The sterol content modulates kinetic and thermodynamic parameters.High temperature promotes the rate of superoxide production. The key element of this enhancement is related to membrane properties such as thickness and viscosity and not to protein structural changes. Membrane viscosity that can be driven by sterols is a paramount parameter of Ea of NADPH oxidase activity. The membrane bilayer state modulated by its sterol content may be considered locally as an enzyme regulator. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.