The property and structure of spread films of meibum extruded from rabbit eyelids and its fractions were investigated using the Langmuir film balance technique and Brewster angle microscopy in order to understand the influence of endogenous ingredients in meibum on the structure and stability of the tear film lipid layer against mechanical stimulus. Surface pressure (pi)-film area (A) isotherms for meibum were measured upon repetitive high-speed compression-expansion cycles and were found almost identical to each other with very small hysteresis, indicating the high stability of the meibum film. Brewster angle microcopy observation implied the spontaneous formation of condensed-phase network structures which consist primarily of wax esters and cholesteryl esters as nonpolar ingredients, coexisting with a monolayer phase of polar lipids two-dimensionally confined by the networks, which were spontaneously formed in the meibum film. The networks were gathered densely and deformed when the film was laterally compressed with barriers of Langmuir trough, but returned to the dispersed networks when expanded. The influence of temperature and salts dissolved in an aqueous subphase was also investigated. The results indicated that the temperature change (20 and 35 degrees C) induced a difference of surface pressure at the same film areas in rather compressed films, and the presence of salts in the subphase expanded the films. However, the features of isotherm and surface morphology of the film, including their reversibility, were maintained. Phospholipid-removed meibum also formed a stable film, but slight changes were found in the hysteresis and film morphology compared to those in the meibum film. In contrast, in a film of phospholipid- and cholesterol-removed meibum, threedimensional aggregates grew upon the first compression and not redispersed by the subsequent expansion, giving noticeable hysteresis between the isotherms. It is considered that high deformability upon compression and resilience upon expansion of the networks as well as reversible collapse and spreading property of the confined monolayer phase would hold the stability of the meibum film against repeated compressions.