Two multi-channel defect-free high-silica MFI membranes have been tested for the removal of ethanol from a 7 mol% and 3 mol% aqueous solution under pervaporation conditions. One membrane is detemplated by calcination at 773 K, and one is ozonicated at 473 K. The flux and ethanol separation factor were determined at 348 K, 360 K, and 373 K. The ethanol separation factors of both membranes were similar and ranged from at 373 K to at 348 K. The ethanol and water fluxes through both membranes in this temperature range are proportional to the component fugacity difference over the membranes. The calcined membrane showed an 80% higher flux than the ozonicated membrane, showing the ozonication could not completely remove the template. After the ozonicated membrane was calcined at 723 K, the flux increased to a similar value as the initially calcined membrane, while the separation factor did not change. Both membranes showed an increase in flux by a factor of two after a subsequent calcination at 823 K, also without affecting the separation factor. The high calcination temperature required for complete detemplation indicates the presence of aluminum in the zeolite framework of the membrane. The membrane stability was monitored for 40 h and 177 h of operation for the initially calcined and ozonicated membrane, respectively. At 348 K the membranes showed stable performance, but the stability of the membranes at elevated temperatures is limited, showing a decrease in both flux and selectivity during operation at 373 K. The effect of the exposure to a water/ethanol mixture on the zeolite membrane is investigated by permporometry measurements of the membranes and by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, magic angle spinning nuclear magnetic resonance (MAS NMR), thermogravimetric analysis (TGA), and nitrogen adsorption experiments using several MFI-type zeolite powders. The most plausible mechanism causing the instability of the membrane is ethoxy formation of the ethanol with the silanol groups and Brønsted acid sites in the zeolite framework combined with the formation of hydrophilic extra-framework pathways by hydrolysis.