When cells are exposed to an external source of H2O2, the rapid enzymatic consumption of H2O2 inside the cell provides the driving force for the formation of the gradient across the plasma and other subcellular membranes. By using the concepts of enzyme latency, the following gradients - formed after a few seconds following the exposure to H2O2 - were estimated in Jurkat T-cells: [H2O2](cytosol)/[H2O2](peroxisomes)=3; [H2O2](extracellular)/[H2O2](cytosol)=7. The procedure presented in this work can easily be applied to other cell lines and provides a quantitative framework to interpret the data obtained when cells are exposed to an external source of H2O2.