Different cathode configurations with carbon paper (CP) and carbon cloth (CC) gas diffusion layer (GDL) are combined with Nafion (R) 115 for GOx enzyme fuel cells. Catalyst-coated Nafion membrane (without a GDL) has resulted with highest impedance and lowest polarization response. Hot pressing of cathode gas diffusion electrode (GDE) onto the Nafion or physical placement onto the alternative chitosan membrane has reduced interface resistance 10 times compared with no-GDL case. The best performance for the Nafion membrane is 2.1 mA cm(-2) current density with hot-pressed CC-GDE. Smallest high-frequency electrolyte (0.55 ohm) and charge transfer resistances (0.15 ohm) were measured with physically placed CC-GDE on the alternative chitosan membrane resulting in maximum short current density of 3 mA cm(-2) with a 4-cm(2) single enzyme fuel cell.