Silicalite membrane preparation conditions related to membrane separation properties were studied focusing on in situ crystallization to prepare highly selective silicalite membranes on porous tubular supports by a single hydrothermal treatment. The membranes morphologies prepared under typical synthesis conditions were characterized by scanning electron microscopy. The silicalite membrane, well-oriented with c axis normal to the support surface, could be prepared by in situ crystallization and a seeding method. The membrane separation properties were controlled by synthesis conditions such as seeding, temperatures, supports, and silica sources. Under certain conditions, such factors as tube pore size, played an important role. Silicalite membranes would be prepared better by in situ crystallization than by a seeding method under the same conditions. For example, the highest ethanol/water separation factor of 106 with a flux of 0.9 kg/m2·h for a feed concentration of 5 wt. % ethanol at 60°C was obtained for the silicalite membrane prepared on an unseeded mullite tube at 175°C for 16 h with colloidal silica, whereas the membranes prepared on the silicalite-seeded tubes had separation factors up to 70. The higher separation selectivity for ethanol over water would be attributed to reduction in nonzeolitic pores, supported by gas permeations of n-butane and i-butane.