Reference cells are used to distinguish between polarization losses of the anode electrode and the cathode electrode in an electrochemical system. In the case of a direct methanol fuel cell (DMFC), a reference electrode can either be attached using a salt bridge or be printed directly on the same ionomer as the working electrode. Several effects influence the reference readings in this setup by disturbing the symmetrical potential distribution of the working electrode inside the membrane. Precise alignment of the electrodes, for example, is required to prevent boundary effects. Thus, a new preparation technique for improved alignment by laser ablation of the catalyst layer is introduced. Results of this segmentation method are validated microstructurally by scanning electron microscopy and energy-dispersive X-ray analysis. It is shown that the relative error in distinguishing anode polarization losses using the reference cell compared to the true value for such a postprocessed membrane electrode assembly is well below 1%. Various influencing factors that cause deviations in the symmetric potential distribution are studied systematically. A simple 2D DMFC model is developed to support the experimental conclusion and to study additional effects. Finally, losses of anode potential derived from half-cell measurements are compared to losses of anode potential derived from reference readings and the results are discussed. (c) 2007 The Electrochemical Society.