The mechanism of direct electron transfer in multicopper oxidases is not well understood. In this work, the mechanism of oxygen reduction in Bilirubin oxidase (BOD) is analyzed using a rotating ring-disc electrode (RRDE). The glassy carbon disc potential was swept from 0.8 V to 0 V while the platinum ring potential was held at 0.8 V. resulting currents were measured. Minimal hydrogen peroxide evolution from BOD is detected on the Pt ring, independent of rotation rate. The electron transfer rate constant, Ice, is calculated to be 1.14 x 10(-3) cm/s. The number of electrons transferred per molecule of oxygen is calculated to be 3.92 electrons using Koutecky-Levich equation and 3.7 +/- 0.2 electrons using mass/charge balance which corresponds to 7.5 +/- 5% of oxygen reduction via the 2 electron pathway with a hydrogen peroxide intermediate. The 4 electron transfer mechanism is preferred because it is more efficient than the two electron mechanism. (C) 2011 Elsevier Ltd. All rights reserved.