The effect of MEA preparation on the performance of single-PEM fuel cells operated at sub-freezing temperatures is presented. The cell performance and durability are dependent on the MEA and are probably influenced by the porosity of the catalyst layers. When a cell is operated isothermally at -10 o C in constant current mode, the voltage gradually decreases over time and eventually drops to zero. AC impedance analysis indicated that the rate of voltage loss is initially due to an increase in the charge transfer resistance and is gradual. After a period, the rate of decay accelerates rapidly due to mass transport limitations at the catalyst and/or gas diffusion layers. The high frequency resistance also increases over time during the isothermal operation at sub-freezing temperatures and was a function of the initial membrane water content. LANL prepared MEAs showed very little loss in the catalyst surface area with multiple sub-freezing operations, whereas the commercial MEAs exhibited significant loss in cathode surface area with the anode being unaffected. These results indicate that catalyst layer ice formation is influenced strongly by the MEA and is responsible for the long-term degradation of fuel cells operated at sub-freezing temperatures. This ice formation was monitored using neutron radiography and was found to be concentrated near cell edges at the flow field turns.