Ultrathin Films of behenic acid methyl ester (BAME) deposited by spin coating on solid substrates or spread at the air/water interface were studied by infrared spectroscopy and Brewster angle microscopy. Anisotropic optical constants of BAME were determined from a transmittance spectrum at normal incidence and a parallel-polarized reflectance spectrum at grazing incidence. A transmittance spectrum recorded at oblique incidence (60degrees) was used to validate these optical constants. The anisotropic extinction coefficients and the polarized ATR spectra were used to calculate the tilt angle of several transition moments and also the molecular tilt angle of BAME molecules assuming an all-trans conformation of the alkyl chain. The results indicate that the alkyl chain of the fatty acid ester is tilted and that the molecular tilt angle is close to 30degrees. PM-IRRAS spectra of BAME at the air/water interface were also recorded as a function of the surface pressure. The splitting of the methylene bending mode shows that the all-tans alkyl chains are packed in a quasi-crystalline structure at the air/water interface, even at low surface pressure. This Finding was further confirmed by Brewster angle microscopy. Furthermore, the band progression due to the methylene wagging modes was observed for the first time in infrared spectra of a Langmuir monolayer. The simulation of the PM-IRRAS spectrum of a BAME monolayer recorded at 30 mN/m using the optical constants obtained from films prepared by spin coating indicate that, as opposed to the orientation on solid substrates, the alkyl chain of BAME is nearly perpendicular to the air/water interface in Langmuir films.