Multilayer polyimide (PI) films were successfully fabricated using the Langmuir-Blodgett (LB) technique. These films were studied in several ways relevant to their use as liquid crystal alignment layers. (1) The influence of dipping speed and creep time on the orientational order of PI-LB films was investigated by means of birefringence measurements. (2) For comparison, we measured the pretilt angles and polar anchoring strengths of liquid crystal (LC) cells assembled with PI-LB films and rubbed PI-LB films as alignment layers. (3) The anchoring direction of the liquid crystal was found to be solely determined by the dipping direction of uppermost PI-LB layer, regardless of the dipping direction of deeper layers. (4) Combining results of the above studies with measurements of pretransitional birefringence (above the isotropic to nematic (I --> N) transition) and observations of the growth of the alignment texture just below the nematic-isotropic transition, we draw the conclusion that the range of interaction between the PI-LB film and LC molecules is quite short (similar to 4.5 Angstrom) and that the alignment mechanism is epitaxial. Therefore, anisotropic short range molecular interactions are responsible for the alignment of the first liquid crystal layer. (5) From studies of PI-LB films deposited perpendicular to the rubbing direction of underlying spin-coated PI films, we also found evidence, as expected, that the grooves induced by the rubbing process are not decisive for LC alignment on a rubbed polymer surface.