A RuO2 diffusion layer is examined for use in direct methanol fuel cells (DMFC) by comparison with acetylene black and Vulcan XC-72R. In the test with a DMFC unit cell, the RuO2 diffusion layer is superior to the other two materials. The difference in performance is interpreted in terms of structural and electrical properties which are evaluated by porosity, scanning electron microscopy and resistance measurements. The RuO2 diffusion layer displays different behaviors at the anode and cathode sides. These characteristics can be attributed to a reduced loss of catalyst in the active catalyst layer, which leads to increased methanol diffusion at the anode and prevention of water flooding in the cathode. The effect of the RuO2 diffusion layer on cell performance becomes more pronounced at lower temperatures and during operation in the presence of air. Finally, a carbon-RuO2 composite is evaluated as a diffusion layer material for a DMFC.