Taking into account published contradictory results concerning the regulation of fatty acid synthase (Fas) by H2O2, we carried out a systematic study where two methods of H2O2 delivery (steady-state and bolus addition) and the effect of a wide range of H2O2 concentrations were investigated. A decrease in Fas activity was observed for cells exposed to 100 and 150 lMH2O2 in a steady-state, while a bolus addition of the same H2O2 concentrations did not alter Fas activity. Similar results were observed for the mRNA levels of FAS1, the gene that encodes Fas subunit b. However, the exposure to a steady-state 50 lMH2O2 dose lead to an increase in FAS1 mRNA levels, showing a biphasic modulation of Fas by H2O2. The results obtained emphasize that cellular effects of H2O2 can vary over a narrow range of concentrations. Therefore, a tight control of H2O2 exposure, which can be achieved by exposing H2O2 in a steady-state, is important for cellular studies of H2O2-dependent redox regulation.