The influence of catalytic layer location on the performance of an open-cathode direct ethanol fuel cell (DEFC) was investigated using three different manufacturing methods for the membrane electrode assembly (MEA). The catalyst loading on both anode and cathode electrodes was 1 mgPtcm-2. In MEA1, the anodic and cathodic catalyst layers (CL) were deposited directly onto the Nafion® membrane surface. MEA2 consisted of two CLs: An inner CL placed on the membrane surface and an outer CL located on the carbon cloth diffuser layer (DL). MEA3 was prepared using Pt-Black as the inner CL and PtRu/C or PtSn/C as the anode outer CL (MEA3a and MEA3b respectively). The combination of an inner and outer CL improved cell performance. Additionally, the incorporation of Pt black as the inner CL in MEA3 diminished ethanol crossover and improved DEFC performance through a significant decrease in mixed potential effects. Furthermore, we report the performance of an open-cathode DEFC using two operational approaches: Air self breathing (ASB) and forced-air convection (FAC). The influence of air supply on DEFC performance was investigated via galvanostatic electrochemical impedance spectroscopy.