Thin (2–7 μm) polycrystalline randomly-oriented zeolite MFI membranes were prepared on an α-Al2O3 support by one or more subsequent hydrothermal treatments. Different particle sizes (275–700 nm) in the layer were achieved by changing the synthesis temperature (371–459 K). Zeolite membranes, prepared by two subsequent hydrothermal treatments have an optimum quality. Membranes prepared by a single hydrothermal treatment have defects and zeolite membranes obtained by three or more hydrothermal treatments become too thick and crack during the removal of the template. The zeolite MFI membranes were characterised by permeation measurements using single gases and mixtures of n-butane (n-C4H10) and iso-butane (i-C4H10). Permselectivities, Fα, and separation factors, α, for n-C4H10/i-C4H10 decrease strongly with an increasing number of defects and hence provide a good indication of membrane quality. The separation, on the other hand, of a CH4/n-C4H10 mixture at high occupation of n-C4H10 is not much affected by small defects. This is ascribed to blocking of those defects by capillary condensation of the butane. Flux values decrease linearly with increasing zeolite crystallite size in the top layer indicating that the effective membrane thickness is only one particle thick. During hydrothermal treatment zeolite particles also grow at the other side of the support but do not decrease significantly the flux through the supported zeolite membrane.