The peripheral Escherichia coli pyruvate oxidase (Pox) was incorporated into a mixed [(alkanethiols + disulfides)/phospholipids] bilayer, supported on a plane gold electrode. The activity of the enzyme was measured by the coupling between enzymatic and electrochemical reactions with ferrocenemethanol in solution (enzymatic electrocatalysis). Cyclic voltammetry allowed the rapid determination of the kinetic parameters of the membrane enzyme in situ. The experimental kinetic data, obtained by cyclic voltammetry, were well-correlated to a kinetic simulation, using a model assuming a ping-pong mechanism for Pox. The apparent Michaelis constants of Pox for pyruvate and ferricinium methanol and the constant for the rate of oxidation of Pox by ferricinium methanol were estimated.