Although biofuel cell research has progressed over the past decade, there were still problems with employing enzymes at air-breathing cathodes, because enzymes need to remain hydrated and most enzyme reactions occur in solution and not in gas phase. This research details an approach to the development of an air-breathing biocathode employing direct electron transfer. This laccase biocathode is studied in two different fuel cell configurations: a proton exchange membrane hydrogen/air fuel cell and a direct methanol fuel cell (DMFC) with an anion exchange membrane. The laccase from the Rhus vernificera biocathode with an enzyme loading of 0.224 mg/cm(2) provides fuel crossover tolerance and provides a high operational current density of 50.0 mA/cm(2) and a maximum power density of 8.5 mW/cm(2) in a 40% methanol DMFC. The laccase biocathode shows a lifetime of 290 h in a DMFC. The hydrogen/air fuel cell provides a stable current for a total of 350 discontinuous hours when operated for 8 h daily.