We investigated the effect of the conditioning methods on improving the direct methanol fuel cell (DMFC) performance. The DMFC performance after the conditioning was measured using a newly developed single cell having an Ag/Ag2SO4 reference electrode, which is not influenced by methanol. As a result, we succeeded in developing an original two-step conditioning method in which the conditioning by fueling H-2 gas is conducted prior to a conventional DMFC conditioning. The anode and cathode characteristics after the two-step conditioning were measured with respect to a reference electrode. Based on the obtained i-E curves, the two-step conditioning is found to improve the methanol oxidation performance at the anode and also suppress the decline of the O-2 reduction performance at the cathode. The high DMFC performance based on the two-step conditioning is well explained by the anode and cathode characteristics. (C) 2009 Elsevier B.V. All rights reserved.