Monolayer formation and Langmuir-Blodgett (LB) film fabrication of bilirubin dihexadecyl ester, H2BE, at an air-water interface on subphases with different pH values and subphases containing different metal ions were investigated. Some information regarding the packing density in monolayers, molecular orientation and complexation by metal ions has been obtained. At the air-water interface the amphiphilic H2BE with its complexing head group in contact with the aqueous phase can form metal complexes with many metal ions present in the aqueous phase. The formation of H2BE-metal complexes leads to changes in the area per H2BE molecule and in the collapse pressure, as well as to changes in the X-ray photoelectron spectroscopy and UV-visible absorption spectra of the monolayer assemblies. Low-angle X-ray diffraction indicates that the LB films of H2BE were Y-type with bilayer spacing of ca. 5.2 nm.