Pyruvate oxidase is a flavoprotein dehydrogenase isolated from Escherichia coli which catalyzes the oxidative decarboxylation of pyruvate to acetate and CO2. In vivo, the enzyme can bind to the bacterial membrane and reduce ubiquinone-8, feeding electrons into the respiratory chain. The purified enzyme has been shown previously to bind to phospholipids and detergents and, upon doing so, is activated. The turnover with ferricyanide as an electron acceptor increases 20- to 30-fold upon lipid binding. In this work, initial velocity and stop-flow kinetics are used to investigate the activation of this enzyme. It is shown that the unactivated form of the enzyme is markedly hysteretic. Progress curves at low substrate concentrations show an initial acceleration in enzyme turnover. This is consistent with the results of stop-flow experiments. Rates obtained for either the reduction of the unactivated flavoprotein by pyruvate or its reoxidation by ferricyanide in single turnover experiments are much slower than the rates predicted by observed turnover in initial velocity studies, in some cases by more than 2 orders of magnitude. The data are best explained by the slow interconversion between two forms of the enzyme, one with low turnover and one which rapidly turns over. As isolated, the enzyme is highly unreactive, as revealed by the stop-flow experiments. During turnover, even in the absence of lipid activators, some of the enzyme converts to the rapid-turnover form. This slow interconversion is shown by kinetic simulation to preclude a steady state from being established. Lipid activators appear to shift the equilibrium to favor the rapid-turnover form of the enzyme. Once the enzyme is "locked" into an activated conformation, the hysteresis is no longer observed, and the stop-flow results are in agreement with data obtained from initial velocity experiments. Activation appears to result in both increased rates of electron transfer into and out of the flavin.