The cytochrome bo3 quinol oxidase from Vitreoscilla (vbo3) catalyses oxidation of ubiquinol and reduction of O2 to H2O. Data from earlier studies suggested that the free energy released in this reaction is used to pump sodium ions instead of protons across a membrane. Here, we have studied the functional properties of heterologously expressed vbo3 with a variety of methods. (i) Following oxygen consumption with a Clark-type electrode, we did not observe a measurable effect of Na+ on the oxidase activity of purified vbo3 solubilized in detergent or reconstituted in liposomes. (ii) Using fluorescent dyes, we find that vbo3 does not pump Na+ ions, but H+ across the membrane, and that H+-pumping is not influenced by the presence of Na+. (iii) Using an oxygen pulse method, it was found that 2 H+/e- are ejected from proteoliposomes, in agreement with the values found for the H+-pumping bo3 oxidase of Escherichia coli (ecbo3). This coincides with the interpretation that 1 H+/e- is pumped across the membrane and 1 H+/e- is released during quinol oxidation. (iv) When the electron transfer kinetics of vbo3 upon reaction with oxygen were followed in single turnover experiments, a similar sequence of reaction steps was observed as reported for the E. coli enzyme and none of these reactions was notably affected by the presence of Na+. Overall the data show that vbo3 is a proton pumping terminal oxidase, behaving similarly to the Escherichia coli bo3 quinol oxidase.